Anna Norberg

Congenital dyserythropoietic anemia type III (CDA III) is caused by a mutation in kinesin family member, KIF23

Haplotype analysis and targeted next-generation resequencing allowed us to identify a mutation in the KIF23 gene and to show its association with an autosomal dominant form of congenital dyserythropoietic anemia type III (CDA III). The region at 15q23 where CDA III was mapped in a large Swedish family was targeted by array-based sequence capture in a female diagnosed with CDA III and her healthy sister. Prioritization of all detected sequence changes revealed 10 variants unique for the CDA III patient. Among those variants, a novel mutation c.2747C>G (p.P916R) was found in KIF23, which encodes mitotic kinesin-like protein 1 (MKLP1). This variant segregates with CDA III in the Swedish and American families but was not found in 356 control individuals. RNA expression of the 2 known splice isoforms of KIF23 as well as a novel one lacking the exons 17 and 18 was detected in a broad range of human tissues. RNA interference-based knock-down and rescue experiments demonstrated that the p.P916R mutation causes cytokinesis failure in HeLa cells, consistent with appearance of large multinucleated erythroblasts in CDA III patients. We conclude that CDA III is caused by a mutation in KIF23/MKLP1, a conserved mitotic kinesin crucial for cytokinesis.

Nerve growth factor R221W responsible for insensitivity to pain is defectively processed and accumulates as proNGF.

We have previously identified a homozygous missense (R221W) mutation in the NGFB gene in patients with loss of deep pain perception. NGF is important not only for the survival of sensory neurons but also for the sympathetic neurons and cholinergic neurons of the basal forebrain; however, it is the sensory neurons that are mainly affected in patients with mutant NGFB. In this report, we describe the effects of the mutation on the function of NGF protein and the molecular mechanisms that may underlie the pain insensitivity phenotype in these patients. We show that the mutant NGF has lost its ability to mediate differentiation of PC12 cells into a neuron-like phenotype. We also show that the inability of PC12 cells to differentiate is due to a markedly reduced secretion of mature R221W NGF. The R221W NGF is found mainly as proNGF, in contrast to wild-type NGF which is predominantly in the mature form in both undifferentiated and differentiated PC12 cells. The reduction in numbers of sensory fibers observed in the patients is therefore probably due to loss of trophic support as a result of drastically reduced secretion of NGF from the target organs. Taken together, these data show a clear decrease in the availability of mutant mature NGF and also an accumulation of proNGF in both neuronal and non-neuronal cells. The differential loss of NGF-dependent neurons in these patients, mainly affecting sensory neurons, may depend on differences in the roles of mature NGF and proNGF in different cells and tissues.

Founder mutations characterise the mutation panorama in 200 Swedish index cases referred for Long QT syndrome genetic testing

BackgroundLong QT syndrome (LQTS) is an inherited arrhythmic disorder characterised by prolongation of the QT interval on ECG, presence of syncope and sudden death. The symptoms in LQTS patients are highly variable, and genotype influences the clinical course. This study aims to report the spectrum of LQTS mutations in a Swedish cohort.MethodsBetween March 2006 and October 2009, two hundred, unrelated index cases were referred to the Department of Clinical Genetics, Umeå University Hospital, Sweden, for LQTS genetic testing. We scanned five of the LQTS-susceptibility genes (KCNQ1, KCNH2, SCN5A, KCNE1, and KCNE2) for mutations by DHPLC and/or sequencing. We applied MLPA to detect large deletions or duplications in the KCNQ1, KCNH2, SCN5A, KCNE1, and KCNE2 genes. Furthermore, the gene RYR2 was screened in 36 selected LQTS genotype-negative patients to detect cases with the clinically overlapping disease catecholaminergic polymorphic ventricular tachycardia (CPVT).ResultsIn total, a disease-causing mutation was identified in 103 of the 200 (52%) index cases. Of these, altered exon copy numbers in the KCNH2 gene accounted for 2% of the mutations, whereas a RYR2 mutation accounted for 3% of the mutations. The genotype-positive cases stemmed from 64 distinct mutations, of which 28% were novel to this cohort. The majority of the distinct mutations were found in a single case (80%), whereas 20% of the mutations were observed more than once. Two founder mutations, KCNQ1 p.Y111C and KCNQ1 p.R518*, accounted for 25% of the genotype-positive index cases. Genetic cascade screening of 481 relatives to the 103 index cases with an identified mutation revealed 41% mutation carriers who were at risk of cardiac events such as syncope or sudden unexpected death.ConclusionIn this cohort of Swedish index cases with suspected LQTS, a disease-causing mutation was identified in 52% of the referred patients. Copy number variations explained 2% of the mutations and 3 of 36 selected cases (8%) harboured a mutation in the RYR2 gene. The mutation panorama is characterised by founder mutations (25%), even so, this cohort increases the amount of known LQTS-associated mutations, as approximately one-third (28%) of the detected mutations were unique.

A mutation in the H/ACA box of telomerase RNA component gene ( TERC ) in a young patient with myelodysplastic syndrome

BackgroundTelomeres are repeated sequences (the hexanucleotide TTAGGG in vertebrates) located at chromosome ends of eukaryotes, protecting DNA from end joining or degradation. Telomeres become shorter with each cell cycle, but telomerase, a ribonucleoprotein complex, alleviates this attrition. The telomerase RNA component (TERC) is an essential element of telomerase, serving as a template for telomere elongation. The H/ACA domain of TERC is indispensable for telomere biogenesis. Mutations in the telomerase components allow accelerated telomere loss, resulting in various disease manifestations, including bone marrow failure. To date, this is the first detailed report of an H-box mutation in TERC that is related to human disease.Case presentationA 26-year-old man with myelodysplastic syndrome (MDS) had very short telomeres. Sequencing identified a single heterozygous mutation in the H box of the patient’s TERC gene. The same mutation was also present in his father and his son, demonstrating that it was germline in origin. The telomere length in the father’s blood was shorter compared to age-matched healthy controls, while it was normal in the son and also in the sperm cells of the patient. In vitro experiments suggested that the mutation was responsible for the telomere shortening in the patient’s leukocytes and contributed to the pathogenesis of bone marrow failure in our patient.ConclusionWe analyzed a mutation (A377G) in the H box of TERC in a young MDS patient who had significantly short-for-age telomeres. As telomeres protect chromosomes from instability, it is highly plausible that this genetic lesion was responsible for the patient’s hematological manifestations, including marrow failure and aneuploidy in the hematopoietic stem cell compartment.

Exclusion of the juvenile myoclonic epilepsy gene EFHC1 as the cause of migraine on chromosome 6, but association to two rare polymorphisms in MEP1A and RHAG

Migraine is a complex, multifactorial disorder for which several loci have been identified in the human genome. We have previously reported linkage to a 10 Mb-region on chromosome 6p12.2-p21.1 in one large Swedish pedigree involving migraine with and without aura. To further investigate this candidate region, a dense set of single nucleotide polymorphic (SNP) markers was used for fine-mapping, decreasing the critical region to 8.5 Mb. Within this region, EFHC1 was recently identified as the disease gene for juvenile myoclonic epilepsy. Migraine and epilepsy has been suggested to share disease mechanisms and therefore EFHC1 is an excellent candidate gene for migraine in this family. Mutation analysis of the gene revealed a disease-segregating polymorphism in the promoter. Association analysis of the polymorphism in a case-control material did not support a role for this gene in migraine pathology. We therefore analyzed five additional candidate genes in the disease-critical region, including MEP1A, RHAG, IL17, SLC25A27 and TNFRSF21. In two of these genes, MEP1A and RHAG, we identified two novel polymorphisms associated with the disease haplotype. The combination of these polymorphisms could not be found in any control individuals, suggesting that they might be involved in genetic predisposition to migraine in this family.

Novel mutations in CRB1 and ABCA4 genes cause Leber congenital amaurosis and Stargardt disease in a Swedish family

This study aimed to identify genetic mechanisms underlying severe retinal degeneration in one large family from northern Sweden, members of which presented with early-onset autosomal recessive retinitis pigmentosa and juvenile macular dystrophy. The clinical records of affected family members were analysed retrospectively and ophthalmological and electrophysiological examinations were performed in selected cases. Mutation screening was initially performed with microarrays, interrogating known mutations in the genes associated with recessive retinitis pigmentosa, Leber congenital amaurosis and Stargardt disease. Searching for homozygous regions with putative causative disease genes was done by high-density SNP-array genotyping, followed by segregation analysis of the family members. Two distinct phenotypes of retinal dystrophy, Leber congenital amaurosis and Stargardt disease were present in the family. In the family, four patients with Leber congenital amaurosis were homozygous for a novel c.2557C>T (p.Q853X) mutation in the CRB1 gene, while of two cases with Stargardt disease, one was homozygous for c.5461-10T>C in the ABCA4 gene and another was carrier of the same mutation and a novel ABCA4 mutation c.4773+3A>G. Sequence analysis of the entire ABCA4 gene in patients with Stargardt disease revealed complex alleles with additional sequence variants, which were evaluated by bioinformatics tools. In conclusion, presence of different genetic mechanisms resulting in variable phenotype within the family is not rare and can challenge molecular geneticists, ophthalmologists and genetic counsellors.

Genetic Heterogeneity and Clinical Outcome in a Swedish Family with Retinal Degeneration Caused by Mutations in CRB1 and ABCA4 Genes

Genetic mechanisms underlying severe retinal dystrophy in a large Swedish family presenting two distinct phenotypes, Leber congenital amaurosis and Stargardt disease were investigated. In the family, four patients with Leber congenital amaurosis were homozygous for a novel c.2557C>T (p.Q853X) mutation in the CRB1 gene, while of two cases with Stargardt disease, one was homozygous for c.5461-10T>C in the ABCA4 gene and another was a compound heterozygous for c.5461-10T>C and a novel ABCA4 mutation c.4773+3 A>G. Sequence analysis of the entire ABCA4 gene in patients with Stargardt disease revealed complex alleles with additional sequence variants.Our results provide evidence of genetic complexity causative of different clinical features present in the same family, which is an obvious challenge for ophthalmologists, molecular geneticists and genetic counsellors.

Diagnostics of rare disorders : whole-exome sequencing deciphering locus heterogeneity in telomere biology disorders

BackgroundThe telomere biology disorders (TBDs) include a range of multisystem diseases characterized by mucocutaneous symptoms and bone marrow failure. In dyskeratosis congenita (DKC), the clinical features of TBDs stem from the depletion of crucial stem cell populations in highly proliferative tissues, resulting from abnormal telomerase function. Due to the wide spectrum of clinical presentations and lack of a conclusive laboratory test it may be challenging to reach a clinical diagnosis, especially if patients lack the pathognomonic clinical features of TBDs.MethodsClinical sequencing was performed on a cohort of patients presenting with variable immune phenotypes lacking molecular diagnoses. Hypothesis-free whole-exome sequencing (WES) was selected in the absence of compelling diagnostic hints in patients with variable immunological and haematological conditions.ResultsIn four patients belonging to three families, we have detected five novel variants in known TBD-causing genes (DKC1, TERT and RTEL1). In addition to the molecular findings, they all presented shortened blood cell telomeres. These findings are consistent with the displayed TBD phenotypes, addressing towards the molecular diagnosis and subsequent clinical follow-up of the patients.ConclusionsOur results strongly support the utility of WES-based approaches for routine genetic diagnostics of TBD patients with heterogeneous or atypical clinical presentation who otherwise might remain undiagnosed.

Novel variants in Nordic patients referred for genetic testing of telomere-related disorders

Telomere-related disorders are a clinically and genetically heterogeneous group of disorders characterized by premature telomere shortening and proliferative failure of a variety of tissues. This study reports the spectrum of telomere-related gene variants and telomere length in Nordic patients referred for genetic testing due to suspected telomere-related disorder. We performed Sanger sequencing of the genes TERT, TERC, DKC1, and TINF2 on 135 unrelated index patients and measured telomere length by qPCR on DNA from peripheral blood leukocytes. We identified pathogenic or likely pathogenic variants in 10 index patients, all of which had short telomeres compared to age-matched healthy controls. Six of the 10 variants were novel; three in TERC (n.69_74dupAGGCGC, n.122_125delGCGG, and n.407_408delinsAA) and three in TERT (p.(D684G), p.(R774*), and p.(*1133Wext*39)). The high proportion of novel variants identified in our study highlights the need for solid interpretation of new variants that may be detected. Measurement of telomere length is a useful approach for evaluating pathogenicity of genetic variants associated with telomere-related disorders.

Characterization of an X-chromosome-linked telomere biology disorder in females with DKC1 mutation

Characterization of an X-chromosome-linked telomere biology disorder in females with DKC1 mutation