Kirsten Svenstrup

Unbiased estimation of total β-cell number and mean β-cell volume in rodent pancreas

We describe a method for unbiased assumption‐free estimation of the total number of β‐cells and the mean β‐cell volume in mouse and rat pancreas based on light microscopy. Such a method, which takes advantage of one of the most recent developments in stereology, the fractionator, has not previously been described. It relies on repeated fractionation of the tissue using systematic uniform random sampling combined with an unbiased counting principle. The method was applied to eight BALB/cBom male mice (56 days) and six Lewis/MOL male rats (47 days). In mice, the total number of β‐cells was 1.06±0.07±106 (mean±SEM) per pancreas with a mean β‐cell volume of 1280±17 μm3, while in rats the total β‐cell number was 2.76±0.42±106 per pancreas with a mean β‐cell volume of 1170±65 μm3. Furthermore, the results showed that in both species the biological variability in the total β‐cell volume is due to differences in the number of β‐cells rather than variability of the mean β‐cell volume. The method can be used to give a precise description of number and volume of β‐cells at different ages, and will make it possible to estimate the contributions of hyper/hypotrophia and hyperlhypoplasia to a given induced or spontaneous change in the total β‐cell mass.

Frontotemporal dementia linked to chromosome 3 (FTD‐3) – current concepts and the detection of a previously unknown branch of the Danish FTD‐3 family

Background:  Among patients with onset of dementia below the age of 65 years, frontotemporal dementia (FTD) is the second most prevalent cause, secondary only to Alzheimer’s disease. Recent advances in understanding the heterogeneous genetic background for different clinical and neuropathological entities of FTD have involved identification of several new causative genes.

A novel mutation in the HSPD1 gene in a patient with hereditary spastic paraplegia

AbstractA mutation in the HSPD1 gene has previously been associated with an autosomal dominant form of spastic paraplegia in a French family. HSPD1 encodes heat shock protein 60, a molecular chaperone involved in folding and quality control of mitochondrial proteins. In the present work we have investigated 23 Danish index patients with hereditary spastic paraplegia (HSP) for mutations in the HSPD1 gene. One patient was found to be heterozygous for a c.1381C > G missense mutation encoding the mutant heat shock protein 60 p.Gln461Glu. The mutation was also present in two unaffected brothers, but absent in 400 unrelated Danish individuals. We found that the function of the p.Gln461Glu heat shock protein 60 was mildly compromised. The c.1381C > G mutation likely represents a novel low-penetrance HSP allele.

Clinical and molecular characterization of limb-girdle muscular dystrophy due to LAMA2 mutations

In this study we describe the clinical and molecular characteristics of limb‐girdle muscular dystrophy (LGMD) due to LAMA2 mutations.

NIPA1 mutation in complex hereditary spastic paraplegia with epilepsy.

Background and purpose:  Hereditary spastic paraplegia (HSP) is a group of clinically and genetically heterogeneous neurodegenerative disorders characterized in the ‘pure’ phenotype by progressive spasticity and weakness of the lower limbs. In the ‘complex’ phenotype, additional neurologic symptoms or signs are found. Mutations in the NIPA1 gene have been reported to cause spastic paraplegia type 6 (SPG6) in 10 families. SPG6 is a rare form of autosomal dominantly inherited HSP associated with a pure phenotype; however, in one complex SPG6 family, idiopathic generalized epilepsy (IGE) has been described and in addition, recurrent microdeletions at 15q11.2 including NIPA1 have been identified in patients with IGE. The purpose was to identify NIPA1 mutations in patients with pure and complex HSP.

Postnatal development of beta-cells in rats. Proposed explanatory model.

The previously shown wave of β‐cell apoptosis and the apparent plateau in the β‐cell mass in the third week of life in rats are still unexplained events. Using a novel design‐based stereological method we investigated the postnatal development of the β‐cell population in Sprague‐Dawley rats. The total β‐cell mass increased from postnatal day 4 until day 16, to be followed by a plateau until day 24, after which it increased further. This plateau was caused by β‐cell hypotrophia as well as decreased net β‐cell formation. The β‐cell mass per unit body weight (the relative β‐cell mass) was five times higher at birth compared with the adult constant level that was reached at approximately 24 days of age. We propose an explanatory model for the postnatal development of the β‐cell population in rats. According to this model, β‐cells in the early postnatal period are immature, i.e. are not susceptible to the mechanism that in later life maintains a constant relative β‐cell mass. Within the following weeks the number of mature β‐cells increases, and from approximately day 24 and onwards the β‐cell population is dominated by mature β‐cells that adjust to match the body weight, keeping a constant relative β‐cell mass. Findings of an apoptotic wave, a plateau phase in the total β‐cell mass development, a period with β‐cell hypotrophia, and the disappearance of insulin‐like growth factor II positive β‐cells at postnatal day 21 all fit well in the model.

Sequence variants in SPAST, SPG3A and HSPD1 in hereditary spastic paraplegia.

Hereditary spastic paraplegia (HSP) is a group of clinically and genetically heterogeneous neurodegenerative disorders characterized by progressive spasticity and weakness in the lower limbs. The most common forms of autosomal dominant HSP, SPG4 and SPG3, are caused by sequence variants in the SPAST and SPG3A genes, respectively. The pathogenic variants are scattered all over these genes and many variants are unique to a specific family. The phenotype in SPG4 patients can be modified by a variant in SPAST (p.Ser44Leu) and recently, a variant in HSPD1, the gene underlying SPG13, was reported as a second genetic modifier in SPG4 patients. In this study HSP patients were screened for variants in SPG3A, SPAST and HSPD1 in order to identify disease causing variations. SPAST was sequenced in all patients whereas subsets were sequenced in HSPD1 and in selected exons of SPG3A. SPG4 patients and their HSP relatives were genotyped for the modifying variant in HSPD1. We report six new sequence variants in SPAST including a fourth non synonymous sequence variant in exon 1 and two synonymous changes of which one has been found in a HSP patient previously, but never in controls. Of the novel variants in SPAST four were interpreted as disease causing. In addition one new disease causing sequence variant and one non pathogenic non synonymous variant were found in SPG3A. In HSPD1 we identified a sporadic patient homozygote for the potential modifying variation. The effect of the modifying HSPD1 variation was not supported by identification in one SPG4 family.

SCA28: Novel Mutation in the AFG3L2 Proteolytic Domain Causes a Mild Cerebellar Syndrome with Selective Type-1 Muscle Fiber Atrophy

The spinocerebellar ataxias (SCA) are a group of rare inherited neurodegenerative diseases characterized by slowly progressive cerebellar ataxia, resulting in unsteady gait, clumsiness, and dysarthria. The disorders are predominantly inherited in an autosomal dominant manner. Mutations in the gene AFG3L2 that encodes a subunit of the mitochondrial m-AAA protease have previously been shown to cause spinocerebellar ataxia type 28 (SCA28). Here, we present the clinical phenotypes of three patients from a family with autosomal dominant cerebellar ataxia and show by molecular genetics and in silico modelling that this is caused by a novel missense mutation in the AFG3L2 gene. Furthermore, we show, for the first time, fluorodeoxyglucose-positron emission tomography (FDG-PET) scans of the brain and selective type I fiber atrophy of skeletal muscle of SCA28 patients indicating non-nervous-system involvement in SCA28 as well.

ATXN2 with intermediate-length CAG/CAA repeats does not seem to be a risk factor in hereditary spastic paraplegia.

Hereditary spastic paraplegia (HSP) confines a group of heterogeneous neurodegenerative disorders characterized by progressive spasticity and lower limb weakness. Age of onset is highly variable even in familial cases with known mutations suggesting that the disease is modulated by other yet unknown parameters. Although progressive gait disturbances, lower limb spasticity and extensor plantar responses are hallmarks of HSP these characteristics are also found in other neurodegenerative disorders, e.g. amytrophic lateral sclerosis (ALS). HSP has been linked to ALS and frontotemporal degeneration with motor neuron disease (FTD-MND), since TDP-43 positive inclusions have recently been found in an HSP subtype, and TDP-43 are found in abundance in pathological inclusions of both ALS and FTD-MND. Furthermore, ataxin-2 (encoded by the gene ATXN2), a polyglutamine containing protein elongated in spinocerebellar ataxia type 2, has been shown to be a modulator of TDP-43 induced toxicity in ALS animal and cell models. Finally, it has been shown that ATXN2 with non-pathogenic intermediate-length CAG/CAA repeat elongations (encoding the polyglutamine tract) is a genetic risk factor of ALS. Considering the similarities in the disease phenotype and the neuropathological link between ALS and HSP we hypothesized that intermediate-length CAG/CAA repeats in ATXN2 could be a modulator of HSP. We show that in a cohort of 181 HSP patients 4.9 % of the patients had intermediate-length CAG/CAA repeats in ATXN2 which was not significantly different from the frequencies in a Danish control cohort or in American and European control populations. However, the mean age of onset was significantly lower in HSP patients with intermediate-length CAG/CAA repeats in ATXN2 compared to patients with normal length repeats. Based on these results we conclude that ATXN2 is most likely not a risk factor of HSP, whereas it might serve as a modulator of age of onset.

CYP7B1: novel mutations and magnetic resonance spectroscopy abnormalities in hereditary spastic paraplegia type 5A.

The SPG5A subtype of Hereditary Spastic Paraplegia (HSP) is a rare autosomal recessive neurodegenerative disorder caused by mutations in the CYP7B1 gene, which encodes a steroid cytochrome P450 7α‐hydroxylase. This enzyme provides the primary metabolic route for neurosteroids. Clinically, SPG5A has been characterized as a pure form of HSP with a variable age of onset, but recently a broader spectrum of phenotypes has been described.