Clinical characteristics and proteome modifications in two Charcot-Marie-Tooth families with the AARS1 Arg326Trp mutation.

BACKGROUND: Aminoacyl tRNA-synthetases are ubiquitously-expressed enzymes that attach amino acids to their cognate tRNA molecules. Mutations in several genes encoding aminoacyl tRNA-synthetases, have been associated with peripheral neuropathy, i.e. AARS1, GARS1, HARS1, YARS1 and WARS1. The pathogenic mechanism underlying AARS1-related neuropathy is not known. METHODS: From 2012 onward, all probands presenting at Telemark Hospital (Skien, Norway) with peripheral neuropathy were screened for variants in AARS1 using an "in-house" next-generation sequencing panel. DNA from patient's family members was examined by Sanger sequencing. Blood from affected family members and healthy controls were used for quantification of AARS1 mRNA and alanine. Proteomic analyses were conducted in peripheral blood mononuclear cells (PBMC) from four affected family members and five healthy controls. RESULTS: Seventeen individuals in two Norwegian families affected by Charcot-Marie-Tooth disease (CMT) were characterized in this study. The heterozygous NM_001605.2:c.976C > T p.(Arg326Trp) AARS1 mutation was identified in ten affected family members. All living carriers had a mild to severe length-dependent sensorimotor neuropathy. Three deceased obligate carriers aged 74-98 were reported to be unaffected, but were not examined in the clinic. Proteomic studies in PBMC from four affected individuals suggest an effect on the immune system mediated by components of a systemic response to chronic injury and inflammation. Furthermore, altered expression of proteins linked to mitochondrial function/dysfunction was observed. Proteomic data are available via ProteomeXchange using identifier PXD023842. CONCLUSION: This study describes clinical and neurophysiological features linked to the p.(Arg326Trp) variant of AARS1 in CMT-affected members of two Norwegian families. Proteomic analyses based on of PBMC from four CMT-affected individuals suggest that involvement of inflammation and mitochondrial dysfunction might contribute to AARS1 variant-associated peripheral neuropathy.

A woman in her fifties with chronic muscle weakness. / En kvinne i 50-årene med langvarig muskelsvakhet.

BACKGROUND: Arthrogryposis multiplex congenita (AMC) is a descriptive term that encompasses a group of congenital, aetiologically heterogeneous conditions characterised by multiple joint contractions. CASE PRESENTATION: As a teenager, the index patient was told she had AMC, as did one of her parents. Subsequently, she wondered how her condition might evolve over time, since her affected parent had become wheelchair- dependent. Her history and clinical findings led to genetic testing which identified a causative variant in the COL6A2 gene, revealing an underlying diagnosis of Bethlem myopathy. INTERPRETATION: Adults who have rare monogenic disorders may lack an aetiological diagnosis because of limited access to genetic laboratory testing in the past. Advances in genetic laboratory diagnostics during the last 10−15 years have made testing more widely available. As exemplified by this case, molecular genetic diagnosis may provide benefits such as information concerning prognosis and treatment options.

De Novo Mutations in Protein Kinase Genes CAMK2A and CAMK2B Cause Intellectual Disability.

Calcium/calmodulin-dependent protein kinase II (CAMK2) is one of the first proteins shown to be essential for normal learning and synaptic plasticity in mice, but its requirement for human brain development has not yet been established. Through a multi-center collaborative study based on a whole-exome sequencing approach, we identified 19 exceedingly rare de novo CAMK2A or CAMK2B variants in 24 unrelated individuals with intellectual disability. Variants were assessed for their effect on CAMK2 function and on neuronal migration. For both CAMK2A and CAMK2B, we identified mutations that decreased or increased CAMK2 auto-phosphorylation at Thr286/Thr287. We further found that all mutations affecting auto-phosphorylation also affected neuronal migration, highlighting the importance of tightly regulated CAMK2 auto-phosphorylation in neuronal function and neurodevelopment. Our data establish the importance of CAMK2A and CAMK2B and their auto-phosphorylation in human brain function and expand the phenotypic spectrum of the disorders caused by variants in key players of the glutamatergic signaling pathway.

Clinical exome sequencing ­ Norwegian findings.

BACKGROUND: New DNA-sequencing technology is revolutionising medical diagnostics. Through the use of exome sequencing, it is now possible to sequence all human genes in parallel. This technology has been widely used in research over the last few years and is now also being applied to diagnostics. The aim of this study was to systematically examine initial experiences with diagnostic exome sequencing in Norway. MATERIAL AND METHOD: This is a retrospective observational study of the results of all exome sequencing performed by the Section of Medical Genetics at Telemark Hospital between December 2012 and October 2014, and includes 125 persons in 46 families. The majority of these families were being investigated for a syndrome (n = 35, 76%) or neurological disease (n = 9, 20%). RESULTS: Exome sequencing detected pathogenic sequence variants in 15 of 46 probands, and variants of unknown significance in 12 probands. Of the 100 patients who stated their wishes regarding feedback of any incidental findings, six indicated that they did not wish to receive such information. There were no incidental findings in this study, but neither were such sequence variants actively looked for. INTERPRETATION: Exome sequencing can enable more patients with syndromes or neurological diseases to receive a causal diagnosis, and to receive this diagnosis at an earlier stage. However, the patients in this study were quite highly selected, and the results must therefore be interpreted with caution.

Hereditary peripheral neuropathies diagnosed by next-generation sequencing.

BACKGROUND: Next-generation sequencing (NGS) is a genetic technique used to determine the order of nucleotides in DNA. The technique has proved to be more efficient than the traditional method, Sanger sequencing, for sequencing multiple genes. NGS is now being used to diagnose disorders in which multiple genes are involved. This study has examined whether next-generation sequencing produces a greater number of positive diagnoses than its traditional counterpart in patients with suspected hereditary peripheral neuropathy. MATERIAL AND METHOD: This study is a retrospective review of samples from 103 patients investigated for hereditary peripheral neuropathy, received by Telemark Hospital in the period 2012-14. After exclusion of duplication/deletion of PMP22, 96 samples were analysed by NGS with physical enrichment of 52 hereditary peripheral neuropathy genes. RESULTS: A genetic cause was identified in 35 patients (34%) with peripheral neuropathy, of which 28 (27%) were point mutations identified by NGS. INTERPRETATION: Of the pathogenic point mutations identified in this study, 12 were in genes that would previously have been analysed by Sanger sequencing in our department, whereas 16 were in genes that would not previously have been tested.

HINT1 neuropathy in Norway: clinical, genetic and functional profiling.

BACKGROUND: Autosomal recessive axonal neuropathy with neuromyotonia has been linked to loss of functional HINT1. The disease is particularly prevalent in Central and South-East Europe, Turkey and Russia due to the high carrier frequency of the c.110G > C (p.Arg37Pro) founder variant. RESULTS: In a cohort of 748 Norwegian patients with suspected peripheral neuropathy, we identified two seemingly unrelated individuals, compound heterozygous for a new variant (c.284G > A, p.Arg95Gln) and the most common pathogenic founder variant (c.110G > C, p.Arg37Pro) in the HINT1 gene. Probands presented with motor greater than sensory neuropathy of various onset, accompanied by muscle stiffness and cramps in the limbs. Furthermore, they displayed non-classical symptoms, including pain in the extremities and signs of central nervous system involvement. Haplotype analysis in both patients revealed a common chromosomal background for p.Arg95Gln; moreover, the variant was identified in Swedish carriers. Functional characterization in HINT1-knockout and patient-derived cellular models, and in HNT1-knockout yeast, suggested that the new variant is deleterious for the function of HINT1 and provided mechanistic insights allowing patient stratification for future treatment strategies. CONCLUSION: Our findings broaden the genetic epidemiology of HINT1-neuropathy and have implications for molecular diagnostics of inherited peripheral neuropathies in Scandinavia.

Ichthyosis prematurity syndrome in two Omani siblings, caused by homozygous c.1A > G mutation in the FATP4 gene.

Ichthyosis prematurity syndrome (IPS) is a rare type of syndromic autosomal recessive congenital ichthyosis (ARCI) caused by a mutation in the SLC27A4 gene that encodes the fatty acid transport protein 4 (FATP4), which is responsible for keratinocyte differentiation and skin barrier function. IPS is characterized by a triad of prematurity, perinatal respiratory asphyxia, and thick vernix caseosa-like scales. In this report, we present the clinical and molecular characterization of IPS in two Omani siblings.

MFN2 point mutations occur in 3.4% of Charcot-Marie-Tooth families. An investigation of 232 Norwegian CMT families.

BACKGROUND: Point mutations in the mitofusin 2 (MFN2) gene has been identified exclusively in Charcot-Marie-Tooth type 2 (CMT2), and in a single family with intermediate CMT. MFN2 point mutations are probably the most common cause of CMT2. METHODS: Two-hundred and thirty-two consecutive unselected and unrelated CMT families with available DNA from all regions in Norway were included. We screened for point mutations in the MFN2 gene. RESULTS: We identified four known and three novel point mutations in 8 unrelated CMT families. The novel point mutations were not found in 100 healthy controls. This corresponds to 3.4% (8/232) of CMT families have point mutations in the MFN2 gene. The phenotypes were compatible with CMT1 in two families, CMT2 in four families, intermediate CMT in one family and distal Hereditary Motor Neuropathy (dHMN) in one family. This corresponds to 2.3% of CMT1, 5.5% of CMT2, 12.5% of intermediate CMT and 6.7% of dHMN families have a point mutation in the MFN2 gene. Point mutations in the MFN2 gene is likely to be the fourth most common cause to CMT after duplication of the peripheral myelin protein 22 (PMP22) gene, and point mutations in the Connexin32 (Cx32) and myelin protein zero (MPZ) genes. CONCLUSIONS: The identified known and novel point mutations in the MFN2 gene expand the clinical spectrum from CMT2 and intermediate CMT to also include possibly CMT1 and the dHMN phenotypes. Thus, genetic analyses of the MFN2 gene should not be restricted to persons with CMT2.

The genetic landscape of axonal neuropathies in the middle-aged and elderly: Focus on MME.

OBJECTIVE: To test the hypothesis that monogenic neuropathies such as Charcot-Marie-Tooth disease (CMT) contribute to frequent but often unexplained neuropathies in the elderly, we performed genetic analysis of 230 patients with unexplained axonal neuropathies and disease onset ≥35 years. METHODS: We recruited patients, collected clinical data, and conducted whole-exome sequencing (WES; n = 126) and MME single-gene sequencing (n = 104). We further queried WES repositories for MME variants and measured blood levels of the MME-encoded protein neprilysin. RESULTS: In the WES cohort, the overall detection rate for assumed disease-causing variants in genes for CMT or other conditions associated with neuropathies was 18.3% (familial cases 26.4%, apparently sporadic cases 12.3%). MME was most frequently involved and accounted for 34.8% of genetically solved cases. The relevance of MME for late-onset neuropathies was further supported by detection of a comparable proportion of cases in an independent patient sample, preponderance of MME variants among patients compared to population frequencies, retrieval of additional late-onset neuropathy patients with MME variants from WES repositories, and low neprilysin levels in patients' blood samples. Transmission of MME variants was often consistent with an incompletely penetrant autosomal-dominant trait and less frequently with autosomal-recessive inheritance. CONCLUSIONS: A detectable fraction of unexplained late-onset axonal neuropathies is genetically determined, by variants in either CMT genes or genes involved in other conditions that affect the peripheral nerves and can mimic a CMT phenotype. MME variants can act as completely penetrant recessive alleles but also confer dominantly inherited susceptibility to axonal neuropathies in an aging population.

Registration of Down syndrome in the Medical Birth Registry of Norway: validity and time trends.

OBJECTIVE: To validate Down syndrome registration in the Medical Birth Registry of Norway (MBRN), 2001-2005, and study time trends and geographical differences in Down syndrome prevalence,1967-2005. DESIGN/SETTING: Population-based cohort study, Norway. POPULATION: 2.3 million pregnancies and births registered in the MBRN, 1967-2005. METHODS: We linked data from the MBRN during 2001-2005 with data from Norway's four laboratories of medical genetics. We calculated sensitivity and positive predictive values (PPV) of the MBRN registration overall, and by background variables. Prevalence rates from 1967 to 2005, overall and regional, were presented graphically as smoothed lowess estimates, crude and standardized for maternal age. Time trends were evaluated, adjusting for maternal age by logistic regression. MAIN OUTCOME MEASURES: Sensitivity, PPV, and prevalence rates. RESULTS: Five hundred and seventy-six verified cases of Down syndrome gave a prevalence of 2.0 per 1,000 among 288,213 births and terminations in the MBRN during 2001-2005. Of verified cases, 470 (81.6%) were registered with Down syndrome in the MBRN, while 470 (90.2%) of 521 MBRN-registered cases were verified. Sensitivity was higher in the Northern (93.1%; p=0.005) and Middle (90.6%; p=0.02) region relative the Southern (76.3%), higher for mothers > or =35 years (92.9%) than younger ones (86.1%; p=0.01), and higher for live births (88.8%) relative stillbirths (55.6%; p<0.001). When adjusting for maternal age, there were no significant time trends in prevalence rates from 1967 to 2005. Regional differences over time were found, probably representing reporting differences. CONCLUSIONS: Validity of registration in the MBRN was satisfactory during 2001-2005. Increasing prevalence rates over time were explained by increasing maternal age.

Two novel connexin32 mutations cause early onset X-linked Charcot-Marie-Tooth disease.

BACKGROUND: X-linked Charcot-Marie Tooth (CMT) is caused by mutations in the connexin32 gene that encodes a polypeptide which is arranged in hexameric array and form gap junctions. METHODS: We describe two novel mutations in the connexin32 gene in two Norwegian families. RESULTS: Family 1 had a c.225delG (R75fsX83) which causes a frameshift and premature stop codon at position 247. This probably results in a shorter non-functional protein structure. Affected individuals had an early age at onset usually in the first decade. The symptoms were more severe in men than women. All had severe muscle weakness in the legs. Several abortions were observed in this family. Family 2 had a c.536 G>A (C179Y) transition which causes a change of the highly conserved cysteine residue, i.e. disruption of at least one of three disulfide bridges. The mean age at onset was in the first decade. Muscle wasting was severe and correlated with muscle weakness in legs. The men and one woman also had symptom from their hands. The neuropathy is demyelinating and the nerve conduction velocities were in the intermediate range (25-49 m/s). Affected individuals had symmetrical clinical findings, while the neurophysiology revealed minor asymmetrical findings in nerve conduction velocity in 6 of 10 affected individuals. CONCLUSION: The two novel mutations in the connexin32 gene are more severe than the majority of previously described mutations possibly due to the severe structural change of the gap junction they encode.

[Episodic ataxias]. / Episodiske ataksier.

BACKGROUND: Episodic ataxias (EAs) exist in sporadic and familial forms. They have considerable genetic and clinical heterogeneity. Better understanding of the disorders will hopefully improve management. MATERIAL AND METHODS: This review is based on personal experience and recent literature. RESULTS: EAs are rare autosomal dominant paroxysmic disorders. At present, five forms have been identified. EA 1 is caused by mutations in the potassium channel gene KCNA1 on chromosome 12p13, EA 2 by mutations in the calcium channel gene CACNA1A gene on chromosome 19p13, and EA 5 by mutations in the calcium channel gene CACNB4&beta on chromosome 2q22-q23. Neither gene nor linkage has been identified for EA 3 and 4. As the name indicates, EAs are characterized by paroxystic ataxia. Patients with EA 1 also have interictal myokymia. EAs are characterized by both locus and allelic heterogeneity, since different genes can cause an almost similar phenotype and different mutations in a gene can cause different disorders. Beside EA, mutations in the KCNA1 gene can cause partial epilepsy and myokymia alone, mutations in the CACNA1A gene can cause familial hemiplegic migraine 1 and spinocerebellar ataxia 6, while mutations in the CACNB4&beta4 gene can cause generalized epilepsy and juvenile myoclonic epilepsy. EA can often be efficiently treated with acetazolamide. INTERPRETATION: EAs are rare autosomal dominant disorders caused by mutations in ion-channel genes. The disorders are not life threatening but disabling without treatment or when medical treatment is ineffective or not tolerated.

[Cerebral cavernous malformations]. / Cerebrale kavernøse malformasjoner.

BACKGROUND: Cerebral cavernous malformations exist in sporadic and familial forms. They have considerable genetic and clinical heterogeneity. Better understanding of these disorders may improve management. MATERIAL AND METHODS: This review is based on personal experience and recent literature. RESULTS: Cerebral cavernous malformations are venous malformations that can be detected with gradient echo MRI of the brain. Approximately 0.5% of the general population have the sporadic form with a single or a few cerebral cavernous malformations which mostly are asymptomatic. Those with the familial form usually have several cavernous malformations caused by an autosomal dominant condition. So far, 3 loci have been identified: CCM1 on chromosome 7q, CCM2 on chromosome 7p, and CCM3 on chromosome 3q, occurring in, respectively, approximately 40%, 20% and 40% of the families. CCM1 is caused by a mutation in the KRIT1 gene and CCM2 is caused by a mutation in the MGC4607 gene, while the gene for CCM3 is not yet identified. Mean age at onset is 20-40, but onset can occur at all ages. The most frequent symptoms are seizures, cerebral haemorrhage, chronic headache and focal neurological deficits. Many carriers are, however, asymptomatic. INTERPRETATION: Sporadic cerebral cavernous malformation is often asymptomatic, while the familial form shows phenotypic and genetic heterogeneity. The symptoms are depending on the location of the malformations as well as whether haemorrhage does occur.

Copy number variations in a population-based study of Charcot-Marie-Tooth disease.

Copy number variations (CNVs) are important in relation to diversity and evolution but can sometimes cause disease. The most common genetic cause of the inherited peripheral neuropathy Charcot-Marie-Tooth disease is the PMP22 duplication; otherwise, CNVs have been considered rare. We investigated CNVs in a population-based sample of Charcot-Marie-Tooth (CMT) families. The 81 CMT families had previously been screened for the PMP22 duplication and point mutations in 51 peripheral neuropathy genes, and a genetic cause was identified in 37 CMT families (46%). Index patients from the 44 CMT families with an unknown genetic diagnosis were analysed by whole-genome array comparative genomic hybridization to investigate the entire genome for larger CNVs and multiplex ligation-dependent probe amplification to detect smaller intragenomic CNVs in MFN2 and MPZ. One patient had the pathogenic PMP22 duplication not detected by previous methods. Three patients had potentially pathogenic CNVs in the CNTNAP2, LAMA2, or SEMA5A, that is, genes related to neuromuscular or neurodevelopmental disease. Genotype and phenotype correlation indicated likely pathogenicity for the LAMA2 CNV, whereas the CNTNAP2 and SEMA5A CNVs remained potentially pathogenic. Except the PMP22 duplication, disease causing CNVs are rare but may cause CMT in about 1% (95% CI 0-7%) of the Norwegian CMT families.

Genetic diagnosis of Charcot-Marie-Tooth disease in a population by next-generation sequencing.

Charcot-Marie-Tooth (CMT) disease is the most prevalent inherited neuropathy. Today more than 40 CMT genes have been identified. Diagnosing heterogeneous diseases by conventional Sanger sequencing is time consuming and expensive. Thus, more efficient and less costly methods are needed in clinical diagnostics. We included a population based sample of 81 CMT families. Gene mutations had previously been identified in 22 families; the remaining 59 families were analysed by next-generation sequencing. Thirty-two CMT genes and 19 genes causing other inherited neuropathies were included in a custom panel. Variants were classified into five pathogenicity classes by genotype-phenotype correlations and bioinformatics tools. Gene mutations, classified certainly or likely pathogenic, were identified in 37 (46%) of the 81 families. Point mutations in known CMT genes were identified in 21 families (26%), whereas four families (5%) had point mutations in other neuropathy genes, ARHGEF10, POLG, SETX, and SOD1. Eleven families (14%) carried the PMP22 duplication and one family carried a MPZ duplication (1%). Most mutations were identified not only in known CMT genes but also in other neuropathy genes, emphasising that genetic analysis should not be restricted to CMT genes only. Next-generation sequencing is a cost-effective tool in diagnosis of CMT improving diagnostic precision and time efficiency.

Stem cell marker-positive stellate cells and mast cells are reduced in benign-appearing bladder tissue in patients with urothelial carcinoma.

Survival after invasive bladder cancer has improved less than that of other common non-skin cancers. In many types of malignancy, treatment failure has been attributed to therapy-resistant stem-like cancer cells. Our aim was therefore to determine identities of stem cell marker-positive cells in bladder cancer tissue and to investigate possible associations between these cells and different forms of bladder neoplasia. We investigated tissue from 52 patients with bladder neoplasia and 18 patients with benign bladder conditions, from a cohort that had been previously described with regard to diagnosis and outcome. The samples were analysed immunohistologically for the stem cell markers aldehyde dehydrogenase 1 A1 (ALDH1) and CD44, and markers of cell differentiation. The majority of stem cell marker-positive cells were located in connective tissue, and a smaller fraction in epithelial tissue. Stem cell marker-positive cells exhibiting possible stem cell characteristics included cells in deeper locations of benign and malignant epithelium, and sub-endothelial cells in patients with or without neoplasia. Stem cell marker-positive cells with non-stem cell character included stellate cells, mast cells, endothelial cells, foamy histiocytes, and neurons. Significantly, ALDH1+ stellate cells and ALDH1+ mast cells were reduced in number in stroma of benign-appearing mucosa of bladder cancer patients. The stem cell markers ALDH1 and CD44 label several types of differentiated cells in bladder tissue. ALDH1+ stellate cells and mast cells appear to be reduced in stroma of normal-appearing mucosa of bladder cancer patients, and may be part of a "field effect" in cancer-near areas.

Diagnosis of intraurothelial neoplasia. Interobserver variation and the value of individual histopathologic attributes.

OBJECTIVE: To determine interobserver variation in histopathologic diagnosis of carcinoma in situ (CIS) and dysplasia (collectively intraurothelial neoplasia [IUN]) of the bladder and identify histomorphologic features important for diagnosis. STUDY DESIGN: A total of 272 consecutive bladder tissue samples were re-evaluated blindly by two general pathologists and one uropathologist for IUN. Discrepancies were resolved jointly. Fifteen histopathologic attributes were evaluated for prediction of diagnosis. Followup revealed recurrence and progression rates for each diagnostic category. RESULTS: Thirty-six percent of specimens contained no evaluable flat mucosa; 51% percent of specimens from papillary urothelial neoplasia (PUN) cases showed CIS. General pathologists detected 56-69% of CIS and 8-42% of dysplasia. Histopathologic features most predictive for CIS were nuclear size, variation in nuclear shape, loss of maturation, loss of polarity, and architectural disorder. None of these individually or in combination exceeded general pathologists' diagnostic accuracy. IUN was not predictive of recurrence or progress. CONCLUSION: Using material mostly consisting of flat mucosa gratuitously provided in PUN resection specimens, IUN carries no prognostic value. General histopathologists detect IUN poorly to moderately, and the five most discriminatory histomorphologic features are insufficient for diagnosis. Interobserver agreement for dysplasia is dismal. Absent flat mucosa in PUN resections predicts recurrence.

Simplification of grading papillary urothelial neoplasia using a reduced set of diagnostic features.

OBJECTIVE: To determine whether a reduced set of the histopathologic features used in internationally accepted classifications is capable of accurately grading papillary urothelial neoplasms (PUN). STUDY DESIGN: All surgical specimens from urinary bladders received during a 2-year period were reexamined by an expert uropathologist for assessing the accuracy of original nonexpert PUN grading and staging. Thirteen histopathologic features entailing 32 attributes were evaluated with regard to prediction of expert grade. Patients were followed for 35-59 months (mean, 47). RESULTS: A total of 88 PUN specimens could be analyzed completely including follow-up specimens. Agreement between original and expert grade was 71% for low-grade and 87% for high-grade PUN, with overall kappa = 0.53. The histomorphologic features most predictive of expert grade were architectural disorder, variability of nuclear enlargement, and absence of umbrella cells. Neither individual histomorphologic attributes nor their combinations were as predictive of expert pathologist grade as original diagnoses. CONCLUSION: Improvements in PUN grading and prognostication are not likely to be accomplished by only reducing the number of histomorphologic features currently recommended by the World Health Organization and International Society of Urological Pathology.