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Positional cloning involves first finding linkage between an inherited phenotype (such as a disease) and a DNA marker, followed by the use of a variety of physical and genetic mapping techniques to move from linkage to mutation. If there is a founder effect within a population, crossovers are often rare between the mutation causing the phenotype and closely situated markers and increasing disequilibrium may be observed as the site of the mutation is approached. Standard coefficients of disequilibrium may, however, be insensitive to the relative position of close markers and the mutation, because they depend upon allele frequencies in the normal population compared to those of the founder chromosome. Using cystic fibrosis in European populations as a model system, alternative methods for determining the position of a mutation are discussed. These include haplotype parsimony and three-way interval likelihood analysis. Both methods predict the location of the major CF mutation accurately from a real set of more than 600 European CF chromosomes.

PURPOSE: In Denmark, the occurrence of Leber hereditary optic neuropathy (LHON) has continuously been monitored since 1944. We provide here a summary of 70 years of data collection including registered lines and subjects by the end of 2012.METHODS: Affected individuals were identified from a national register of hereditary eye diseases at the National Eye Clinic (NEC), a tertiary low vision rehabilitation center for the entire Danish population. The assembling of LHON pedigrees was based on the reconstruction of published families and newly diagnosed cases from 1980 to 2012 identified in the files of NEC. Genealogic follow-up on the maternal ancestry of all affected individuals was performed to identify a possible relation to an already known maternal line. A full genotypic characterization of the nation-based LHON cohort is provided.RESULTS: Forty different lines were identified. The number of live affected individuals with a verified mitochondrial DNA mutation was 104 on January 1, 2013, which translates to a prevalence rate of 1:54,000 in the Danish population.CONCLUSIONS: Haplogroup distribution as well as mutational spectrum of the Danish LHON cohort do not deviate from those of other European populations. The genealogic follow-up reveals a relatively high turnover among families with approximately 15 newly affected families per century and the dying out of earlier maternal lines.

We evaluated the diagnostic value of respiratory chain (RC) enzyme analysis of muscle in adult patients with mitochondrial myopathy (MM). RC enzyme activity was measured in muscle biopsies from 39 patients who carry either the 3243A>G mutation, other tRNA point mutations, or single, large-scale deletions of mtDNA. Findings were compared with those obtained from asymptomatic relatives with the 3243A>G mutation, myotonic dystrophy patients, and healthy subjects. Plasma lactate concentration, maximal oxygen uptake, and ragged-red fibers/cytochrome c-negative fibers in muscle were also determined. Only 10% of patients with the 3243A>G point mutation had decreased enzyme activity of one or more RC complexes, whereas this was the case for 83% of patients with other point mutations and 62% of patients with deletions. Abnormal muscle histochemistry was found in 65%, 100%, and 85% of patients, respectively, in these three groups. The results indicate that RC enzyme analysis in muscle is not a sensitive test for MM in adults. In these patients, abnormal muscle histochemistry appears to be a better predictor ofMM.

Udgivelsesdato: 2009-Aug INTRODUCTION: The point mutation of 3243A>G mtDNA is the most frequent cause of mitochondrial diabetes, often presenting as the syndrome maternally inherited diabetes and deafness (MIDD). The mutation may also cause myopathy, ataxia, strokes, ophthalmoplegia, epilepsy, and cardiomyopathy in various combinations. Consequently, it is difficult to predict the "phenotypic risk profile" of 3243A>G mutation-positive subjects. The 3243A>G mutation coexists in cells with wild-type mtDNA, a phenomenon called heteroplasmy. The marked variability in mutation loads in different tissues is the main explanation for the different phenotypes associated with this mutation. AIM: The aim of the study was to screen asymptomatic and oligosymptomatic 3243A>G mtDNA carriers for diabetes and myopathy. METHODS: The study is a case-control study. Nineteen adult 3243A>G carriers presumed to be normoglycemic and matched healthy controls were subjected to an oral glucose tolerance test. Twenty-six adult 3243A>G carriers with unknown myopathy status and 17 healthy controls had a maximal cycle test and a muscle biopsy performed. The mutation loads were quantified in blood and muscle biopsies and correlated to the clinical manifestations of the mutation. RESULTS: In the presumed normoglycemic 3243A>G-positive subjects, one subject had overt diabetes, and 10 subjects had impaired glucose tolerance. Sixteen of the 26 subjects with unknown oxidative capacity fulfilled criteria for myopathy. The mutation load in blood and muscle correlated with the age for diagnosis of impaired glucose homeostasis and hearing impairment (rho = -0.71 to -0.78; P < 0.0001). CONCLUSION: The findings suggest that 3243A>G mutation carriers should be screened for diabetes and myopathy.

We investigated whether mutations in the genes that code for the different subunits of the amiloride-sensitive epithelial sodium channel (ENaC) might result in cystic fibrosis (CF)-like disease. In a small fraction of the patients, the disease could be potentially explained by an ENaC mutation by a Mendelian mechanism, such as p.V114I and p.F61L in SCNN1A. More importantly, a more than three-fold significant increase in incidence of several rare ENaC polymorphisms was found in the patient group (30% vs. 9% in controls), indicating an involvement of ENaC in some patients by a polygenetic mechanism. Specifically, a significantly higher number of patients carried c.-55+5G>C or p.W493R in SCNN1A in the heterozygous state, with odds ratios (ORs) of 13.5 and 2.7, respectively.The p.W493R-SCNN1A polymorphism was even found to result in a four-fold more active ENaC channel when heterologously expressed in Xenopus laevis oocytes. About 1 in 975 individuals in the general population will be heterozygous for the hyperactive p.W493R-SCNN1A mutation and a cystic fibrosis transmembrane conductance regulator (CFTR) gene that results in very low amounts (0-10%) functional CFTR. These ENaC/CFTR genotypes may play a hitherto unrecognized role in lung diseases.

Summary Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder with impaired function of the adrenal cortex caused by mutations in the CYP21A2 gene. Deficiency of steroid 21-hydroxylase accounts for 80–95% of CAH cases. Testicular adrenal rest tumours (TART) may be prevalent in up to 95% of CAH adults and may already appear during childhood. Whether genotype sub-types can account for the development of TART has not been investigated previously. We therefore investigated this by coupling clinical information of CAH patients with information of their genetic mutation. In 49 male patients (age 2.6–40.3 years) with 21-hydroxylase deficiency, testicular ultrasound examinations were performed and CYP21A2 genotypes determined. These were grouped according to the residual 21-hydroxylase activity: group Null (complete enzyme impairment), group A (almost complete enzyme impairment), group B (severe enzyme impairment) and group C (partial impairment). TART were observed in 27 of 49 patients (55%). For the 23 patients younger than 18 years, TART were present in 11 (48%), the youngest patient being 7.5 years old. The presence of TART was dependent on the CYP21A2 genotype: 27 of 37 patients (73%) with the most severe mutations (groups Null and A) had TART, whereas none of 12 patients with the milder mutations (groups B and C) had TART. We conclude that TART were most frequently detected in patients with severe CYP21A2 mutations, and may occur already in early childhood in such patients.

Mitochondrial DNA depletion, encephalomyopathic form, with methylmalonic aciduria is associated with mutations in SUCLA2, the gene encoding a beta subunit of succinate-CoA ligase, where 17 patients have been reported. Mutations in SUCLG1, encoding the alpha subunit of the enzyme, have been reported in only one family, where a homozygous 2 bp deletion was associated with fatal infantile lactic acidosis. We here report a patient with a novel homozygous missense mutation in SUCLG1, whose phenotype is similar to that of patients with SUCLA2 mutations.

A reinvestigation of a Danish family with X-linked inherited congenital nystagmus through 6 generations revealed a congenital stationary retinal dysfunction syndrome with characteristics of both incomplete congenital stationary night blindness and Aland Eye Disease. In spite of rather uniform electrophysiological findings in our patients, this retinal disorder which affects both cones and rods demonstrated considerable intrafamilial diversity with respect to visual acuity, nystagmus, refractive state and fundus pigmentation.

Background and purpose: It is unknown whether prolonged training is a safe treatment to alleviate exercise intolerance in patients with mitochondrial DNA (mtDNA) mutations. Methods: The effect of 3 and 12 months training and 3–12 months deconditioning was studied in four patients carrying different mtDNA mutations. Results: Three-month moderate-intensity training increased oxidative capacity by 23%, which was sustained after 6–12 months of low-intensity training. Training and deconditioning did not induce adverse effects on clinical symptoms, muscle morphology and mtDNA mutation load in muscle. Conclusion: Long-term training effectively improves exercise capacity in patients with mitochondrial myopathy, and appears to be safe.

Over 100 mutations in the myophosphorylase gene, which cause McArdle disease, are known. All these mutations have resulted in a complete block of muscle glycogenolysis, and accordingly, no genotype-phenotype correlation has been identified in this condition. We evaluated physiologic and genetic features of two patients with a variant form of McArdle disease, associated with unusually high exercise capacity. Physiologic findings were compared to those in 47 patients with typical McArdle disease, and 17 healthy subjects. Subjects performed an ischaemic forearm exercise test to assess lactate and ammonia production. Peak oxidative capacity (VO2max) and cardiac output were determined, using cycle ergometry as the exercise modality. The two patients with atypical McArdle disease carried common mutations on one allele (R50X and G205S), and novel splice mutations in introns 3 [IVS3-26A>G (c.425-26A>G)] and 5 [IVS5-601G>A (c.856-601G>A)] on the other allele. Plasma lactate after ischaemic exercise decreased in all typical McArdle patients, but increased in the two atypical McArdle patients (10% of that in healthy subjects). Peak workload and oxidative capacity were 2-fold higher in patients with atypical McArdle disease compared to typical McArdle patients. Oxygen uptake, relative to cardiac output, was severely impaired in the 47 patients with typical McArdle disease, and partially normalized in the milder affected McArdle patients. These findings identify the first distinct genotype-phenotype relationship in McArdle disease, and indicate that minimal myophosphorylase activity ameliorates the typical McArdle disease phenotype by augmenting muscle oxidative capacity. The milder form of McArdle disease provides important clues to the level of functional myophosphorylase needed to support muscle oxidative metabolism.

Summary Mutations in PYGM, encoding the muscle-specific glycogen phosphorylase (myophosphorylase), are responsible for McArdle disease. Among Caucasians, a large proportion of patients are homozygous for the R50X mutation, but other mutations can affect all the 20 exons of PYGM, making mutation detection laborious. We have developed a high-resolution melting (HRM) assay for mutation detection in PYGM. Twelve McArdle patients were investigated, in whom pre-screening had ruled out homozygosity or compound heterozygosity for the two common G205S and R50X mutations. In total, we identified 16 different variations. Thirteen of these are pathogenic, and three were classified as polymorphisms. Nine variations had not previously been described. One of the novel mutations, c.2430C > T, was initially predicted to result in a silent G810G change, but cDNA analysis demonstrated that the mutation led to abnormal mRNA processing. The HRM protocol reduced the need for direct sequencing by approximately 85%, and is a good approach to search for new mutations in PYGM.

A deletion on one chromosome and a mutant allele on the other may cause an autosomal recessive disease. We report on two patients with mental retardation, dysmorphic features and low catalytic activity of arylsulfatase A. One patient had a pathogenic mutation in the arylsulfatase A gene (ARSA) and succumbed to metachromatic leukodystrophy (MLD). The other patient had a pseudoallele, which does not lead to MLD. The presenting clinical features and low arylsulfatase A activity were explained, in each patients, by a deletion of 22q13 and, thereby, of one allele of ARSA.

The multiplex ligation-dependent probe amplification (MLPA) technique is a sensitive technique for relative quantification of up to 50 different nucleic acid sequences in a single reaction, and the technique is routinely used for copy number analysis in various syndromes and diseases. The aim of the study was to exploit the potential of MLPA when the DNA material is limited. The DNA concentration required in standard MLPA analysis is not attainable from dried blood spot samples (DBSS) often used in neonatal screening programs. A novel design of MLPA probes has been developed to permit for MLPA analysis on small amounts of DNA. Six patients with congenital adrenal hyperplasia (CAH) were used in this study. DNA was extracted from both whole blood and DBSS and subjected to MLPA analysis using normal and modified probes. Results were analyzed using GeneMarker and manual Excel analysis. A total number of 792 ligation events were analyzed. In DNA extracted from dried blood spot samples, 99.1% of the results were accurate compared to 99.9% of the results obtained in DNA from whole blood samples. This study clearly demonstrates that MLPA reactions with modified probes are successful and reliable with DNA concentrations down to 0.3 ng/microL (1.6 ng total). This broadens the diagnostic perspectives of samples of DBSS allowing for copy number variation analysis in general and particularly testing for CAH.

An 11-year-old girl sitting in the kitchen suddenly complained of dizziness. Seconds later, she collapsed and had cardiac arrest. Cardiopulmonary resuscitation was initiated immediately but was unsuccessful, despite prolonged efforts. The girl was previously healthy and had never experienced any cardiac or muscular symptoms. At autopsy, the gross examination was normal. The heart weighed 230 g; the chamber sizes, wall thicknesses, and myocardial appearance were normal. Microscopic examination of the right ventricular myocardium showed marked fibrofatty replacement (Figures 1 and 2⇓). No replacements were found in the left ventricular myocardium or in the skeletal musculature. On this basis, it was concluded that the cause of death was arrhythmogenic right ventricular cardiomyopathy (ARVC). Figure 1. Biopsy from the right ventricular free wall showing fibrofatty replacement. Hematoxylin-eosin stain, magnification ×25. …

Background: Mutations in the Presenilin 2 gene (PSEN2) are rare causes of Alzheimer’s disease (AD). Pathogenic mutations in the genes associated with autosomal dominant inherited AD have been shown to alter processing of the amyloid precursor protein (APP) resulting in a relative increase of the amount of Aβ42 peptide. Methods and results: We present a patient with neuropathologically confirmed early-onset AD characterized by profound language impairment. The patient was heterozygous for a novel missense mutation in exon 11 of the PSEN2 gene leading to a predicted amino acid substitution from valine to methionine in position 393, a conserved residue. However, in vitro expression of PSEN2 V393M cDNA did not result in detectable increase of the secreted Aβ42/40 peptide ratio. The mutation was not found in 384 control individuals tested. Conclusions: The possible pathogenic nature of the mutation is not clarified. We discuss the limitations of functional PSEN2 studies and the challenges associated with genetic counselling of family members at risk.

The aim of the current study was to assess the psychological and social impact of delta F508 carrier screening for cystic fibrosis among pregnant women. The impact of carrier screening was assessed in terms of anxiety, perception of health, reproductive decisions, retention of results, and sharing of information with relatives by two self-administered questionnaires sent to 160 women with a positive and 200 women with a negative test result. While no attempt was made to make women accept or decline testing, 22-28% of those tested found it difficult to reject the test when offered. Women with a positive test result became more anxious than did women with a negative result. However, their perception of future health did not change. Most carriers shared the information about their result with relatives and friends. Carriers had the best retention of pretest information and test result, although a third of the carriers believed that they could not have a child with cystic fibrosis when the partner's test for delta F508 and five other mutations were negative. Most women with a negative test result did not remember their result correctly a year after testing. Few women with a positive result changed their reproductive plans because of the result of the test.

The relationship between the cystic fibrosis (CF) genotype and endocrine and exocrine pancreatic function was studied in 215 CF patients. In the 211 patients with the delta F508 mutation, endocrine pancreatic function (oral glucose tolerance; WHO criteria) was normal in 72.5%, impaired in 12.3%, and diabetic in 15.2% of the patients, with no difference between CF patients homozygous (N = 163, median age 15 years, range 2-40) or heterozygous (N = 48, 18 years, 3-40; age difference not significant) for the delta F508 mutation. Exocrine pancreatic sufficiency (no need for pancreatic enzyme substitution) was found in 0.6% of the patients homozygous for the delta F508 mutation and in 10.4% of the heterozygotes (p less than 0.01). Homozygous patients with pancreatic insufficiency took more pancreatic enzyme capsules (median 42 per day, range 0-192) than the heterozygotes (29 per day, 0-300; p less than 0.001). The four patients (1.9%) without the delta F508 mutation had normal glucose tolerance but exocrine pancreatic insufficiency. In conclusion, the major mutation genotype in CF (delta F508) affects the severity of the exocrine pancreatic insufficiency, whereas endocrine pancreatic function is unrelated to this genotype.

Linkage analysis in a family with X-linked myopia gave a positive LOD score (z = 4.8 at theta = 0) for linkage to F8C. These results suggest a provisional assignment for the locus of this syndrome to the distal part of the X chromosome at Xq28. Based on the clinical and genetic evidence, a redefinition of this clinical syndrome, named Bornholm Eye Disease (BED), was made to include amblyopia, myopia, and deuteranopia. Facultative signs were optic nerve hypoplasia, reduced electroretinographic flicker function, and non-specific retinal pigment abnormalities.

Background— Rare mutations in the mitochondrial genome may cause disease. Mitochondrial haplogroups defined by common polymorphisms have been associated with risk of disease and longevity. We tested the hypothesis that common haplogroups predict risk of ischemic cardiovascular disease, morbidity from other causes, mortality, and longevity in a general population of European descent. Methods and Results— We followed 9254 individuals from the Danish general population, in the Copenhagen City Heart Study, prospectively for risk of ischemic cardiovascular disease, morbidity from other causes, and mortality during 25 and 11 years, respectively. Haplogroup frequencies were as follows: H (45.9%), U (15.9%), T (9.9%), J (9.1), K (6.2%), V (4.5%), W/I (3.8%), and Z (3.5%). Hazard ratios for hospitalization due to all cardiovascular disorders (haplogroup U: 1.0 [95% confidence interval{CI}, 0.9 to 1.1]; T: 0.9 [95% CI, 0.8 to 1.0]; J: 1.0 [95% CI, 0.9 to 1.1]; K: 1.0 [95% CI, 0.9 to 1.2]; V: 1.0 [95% CI, 0.9 to 1.2]; W/I: 0.8 [95% CI, 0.7 to 1.0]; Z: 1.0 [95% CI, 0.8 to 1.2]), ischemic heart disease (U: 0.9 [95% CI, 0.8 to 1.1]; T: 0.9 [95% CI, 0.7 to 1.0]; J: 1.1 [95% CI, 0.9 to 1.2]; K: 1.1 [95% CI, 0.9 to 1.3]; V: 1.1 [95% CI, 0.9 to 1.4]; W/I: 1.1 [95% CI, 0.8 to 1.4]; Z: 1.1 [95% CI, 0.8 to 1.4]), and ischemic cerebrovascular disease (U: 1.1 [95% CI, 0.9 to 1.4]; T: 0.9 [95% CI, 0.7 to 1.2]; J: 1.1 [95% CI, 0.9 to 1.4]; K: 1.0 [95% CI, 0.8 to 1.4]; V: 1.1 [95% CI, 0.8 to 1.5]; W/I: 0.8 [95% CI, 0.5 to 1.3]; Z: 0.9 [95% CI, 0.6 to 1.4]) did not differ from 1.0 for any haplogroup versus the most common haplogroup H. Results were similar for hospitalization due to infectious and parasitic diseases, respiratory infections, respiratory disorders, malignant neoplasms, digestive disorders, musculoskeletal disorders, neuropsychiatric disorders, and miscarriages. Likewise, hazard ratios for death from all causes were not different from 1.0 for any haplogroup versus haplogroup H (U: 1.0 [95% CI, 0.8 to 1.1]; T: 0.9 [95% CI, 0.8 to 1.1]; J: 0.9 [95% CI, 0.8 to 1.1]; K: 1.0 [95% CI, 0.8 to 1.2]; V: 1.1 [95% CI, 0.9 to 1.3]; W/I: 0.8 [95% CI, 0.7 to 1.1]; Z: 0.9 [95% CI, 0.7 to 1.2]). Finally, after stratification by major causes of death, hazard ratios remained insignificant. Conclusions— Our results do not support an association of mitochondrial haplogroups with risk of ischemic cardiovascular disease, morbidity from other causes, mortality, or longevity in a large general population of European descent.

Background Approximately 1% of all cases of Alzheimer's disease are inherited autosomal dominantly, and to date, three causative genes have been found, the Presenilin 1 (PSEN1) gene, the Presenilin 2 (PSEN2) gene and the Amyloid precursor protein (APP) gene. We describe atypical phenotypic features in a family with a pathogenic APP gene mutation and discuss possible explanations for these atypical features. Methods and results We report a family with a history of dementia compatible with autosomal dominant transmission. The disease course in the proband was not typical for Alzheimer's disease as the diagnosis was preceded by 8 years of an isolated amnesia. Further, the proband had epilepsy with complex partial seizures and central degenerative autonomic failure as determined by clinical physiology. Sequencing the three known causative Alzheimer genes revealed a pathogenic missense mutation, APP Thr714Ala (the Iranian mutation). Conclusions The atypical clinical phenotype with long prodromal phase, autonomic failure and seizures in this new proband with the APP Thr714Ala mutation illustrates the clinical heterogeneity in families with identical pathogenic mutations.

Udgivelsesdato: 2008-Apr We report clinical, molecular, neuroimaging and neuropathological features of a Danish family with autosomal dominant inherited dementia, a clinical phenotype resembling Alzheimer's disease and a pathogenic mutation (R406W) in the microtubule associated protein tau (MAPT) gene. Pre-symptomatic and affected family members underwent multidisciplinary (clinical, molecular, neuroimaging and neuropathological) examinations. Treatment with memantine in a family member with early symptoms, based on the clinical phenotype and the lack of specific treatment, appears to stabilize the disease course and increase the glucose metabolism in cortical and subcortical areas, as determined by serial [F(18)]FDG-PET scanning before and after initiation of treatment. Neuropathological examination of a second affected and mutation-positive family member showed moderate atrophy of the temporal lobes including the hippocampi. Microscopy revealed abundant numbers of tau-positive neurofibrillary tangles in all cortical areas and in some brainstem nuclei corresponding to a diagnosis of frontotemporal lobe degeneration on the basis of a MAPT mutation. The clinical and genetic heterogeneity of autosomal dominant inherited dementia must be taken into account in the genetic counselling and genetic testing of families with autosomal dominantly inherited dementia in general.

The Twinkle gene product is important for mtDNA replication. Only a few reports have investigated the clinically effect of mutations in this gene. We describe a new de novo mutation (1110C>A) in the PEO1 gene in a mother and her two sons. The mother had progressive ophthalmoplegia, limb weakness, sensory neuropathy, elevated resting plasma lactate, glucose intolerance and impaired VO2max while her sons only had mild ptosis. In accordance with the clinical presentation, abnormal morphological findings in muscle and multiple deletions and depletion of mtDNA in muscle were more pronounced in the proband than in her sons.

A magnificent casket in a woman’s grave from the Viking AgeIn 2004 and 2006, Moesgård Museum excavated 21 Viking Age graves – 16 inhumation graves and five cremation graves – at Haldum Church, 20 km northwest of Århus in Eastern Jutland (fig. 1). All the graves with datable finds are from the 10th century; only one example will be presented here.The grave is a simple inhumation burial. The skeleton had completely disappeared and there were no signs of a coffin (fig. 2). About 40 cm from the western end of the grave base lay 16 glass beads scattered around over an area of about 20 x 30 cm. These comprised one red and one orange bead of opaque glass, one bead of white glass and 13 small beads of yellow glass. In the centre of the grave was a slate whetstone (fig. 3). At the eastern end was a concentration of iron fittings, and it soon became clear that these represented the remains of a casket.During excavation of the fittings a well-preserved lock turned up (fig. 4). One end of the lock’s cover plate terminates in a point with concave flared sides, while the other (broad) end divides into two prongs, between which a transverse, semicircular fitting has rusted fast. An iron band has been mounted across the pointed end; this is possibly a repair. The cover plate curves slightly along its length, which means that the lock must have been fitted on to a curved surface. The back of the lock is covered by a quadrangular plate. The distance between front and back plates is 15 mm, showing the thickness of the material within which the lock was fitted. On removing the back plate the lock construction can be seen; this is of well-known Viking Age type. It operates in such a way that the key – after having been placed into the keyhole – is pushed a little to the side so that it squeezes together the two springs of the bolt. The bolt is hereby disengaged and can be pulled back by means of a slide bar, which sits in continuation of the keyhole (fig. 5).The lock bolt is pushed into the above-mentioned, semicircular fitting. The latter comprises two plates separated by a c. 10 mm wide side piece, which gives the fitting the appearance of a small box. Three fittings of this type were found (fig. 6).Further to these components are edge and corner plates. The edge fittings all have the form of narrow, indented borders with small holes between the points. A fragment is shown lowermost on fig. 6. The corner plates have been bent back around the corner joints of the casket and are pointed at both sides.Certain other fittings were at first inexplicable. These mysterious pieces include three triangular fittings with small slide bars on their upper surface and 30 mm long bolts below (fig. 7). There are also fragments of some zip-like iron bands. These are slightly curved along their length and have a roof-shaped cross-section (fig. 8). Finally, there was a robust rivet with a square head, ingeniously worked with inwardly flared, tapered sides and crowned by a small boss.Wood imprints on the back of the fittings show that the casket was made of oak. The imprints were very useful when reconstructing the casket as they clearly show both the longitudinal direction of the wood and the depressions within it. There were imprints from a textile of coarse linen weave on the exterior of some of the edge fittings.It seems that the casket from Haldum had the same construction as the Bamberg casket (figs. 9 and 10). Each of the four sides of the Bamberg casket has, at its centre, a raised semicircular area covered by fittings. The exterior is decorated with a mask and there is a hole on the inside. The bolts of the lid presumably engaged with these holes when the casket was locked. Carved grooves in the wood under the ornamental plates of the lid lead out by way of the holes into the four semicircles. The semicircular fittings of the Haldum casket are, with regard to size and shape, completely identical to the mask fittings seen on the Bamberg casket. One of them is, as mentioned above, rusted to the lock, the bolt of which has been pushed into a hole on its inner surface. Consequently, its function is clear; it is also clear that the casket was locked when placed in the grave.The lid of the Bamberg casket is divided by ornamental bands into four triangular fields (fig. 11). In one of these fields (A), a T-shaped keyhole is apparent, and in continuation of this there is a slot for a slide bar. In the field opposite (C), there is a small hole and each of other fields (B and D) has a partly damaged slide-bar slot. We are so fortunate that the fittings surviving from the Haldum casket include slide bars, bolts and other lock parts that have been lost from the Bamberg casket.It is possible to place the lock and the various fittings from the Haldum casket in a square of the same dimensions as the lid of the Bamberg casket. In the fields created by arranging the zip-shaped fittings to form a diagonal cross, there is space for the lock and the three triangular fittings (fig. 11). The excavation photo in fig. 12 shows the three types of fittings in their original positions. In continuation of the keyhole, the lock has a small slide bar whereby the bolt was pushed into one of the semicircular fittings (side A). The forks of the lock plate extend down on either side of this fitting, demonstrating that there was a central depression in the four sides of lid in order to accommodate the semicircular fittings, as seen on the Bamberg casket. In the triangular fitting, which was located opposite the lock (side C), there is also a small slide bar but no slot in which it could move. Similarly, the wood imprint on the back shows that there was no depression to allow a bolt to be pushed back and forth. On the corresponding side of the lid of the Bamberg casket, the carved depression for the bolt is less marked than on the other sides. On the two remaining triangular fittings from the Haldum casket, the slide bars are located in 15 mm long slots (sides B and D). On the reverse, clear depressions are seen in the wood imprint in which the bolts were slid back and forth (fig. 13). If the fittings are arranged in this way, all the pieces show the same longitudinal direction of the wood imprints on their reverse. This indicates that the casket lid was made from one piece of wood.As is apparent from the carvings on the Bamberg casket, the slide bars of the closing mechanism were located close to the centre of the lid. The hidden grooves for the bolts run from here, under the ornamental plates, and emerge at the edge of the lid. Apparently, the Haldum casket did not have ornamental plates screening the grooves for the bolts. As a consequence, the triangular fittings with the slide bars were placed close to the edge of the lid so that they met the semicircular fittings. In this way it was only necessary to have short grooves for the bolts, and these were covered by the fittings.The way in which the lid and the casket are fitted to one another, together with the absence of hinges, indicates that the lid was loose and was lifted completely off in order to open the box. The bolt opposite the lock (side C) was permanently pushed forwards and was the first to be pushed into the matching semicircular fitting, after which the lid was tilted down into place. After this, the two bolts at the sides (B and D) were extended to keep the lid fastened. Finally, the lock’s bolt was pushed into place and the casket was then locked.By observing the curvature of the striker plate, the triangular fittings, the zip-shaped fittings and some of the edge fittings, which have a curved cross-section, it is possible to reconstruct the shape of the lid (see fig. 10). Its height was c. 45 mm. The rivet must have marked the centre of the lid, corresponding to the cruciform fitting on the lid of the Bamberg casket.The body of the Bamberg casket was assembled by pushing the end surface of one side against the side surface of the next. The wood imprints on the corner plates of the Haldum casket show that the same technique was also used here. It is apparent from these wood imprints, as well as the distance between front and back of the semicircular fittings, that the sides were about 10 mm thick. The wood imprints on the inner side of the semicircular fittings show that the tree rings on the side pieces ran vertically. Had they run horizontally, this would have rendered these curves a weak point.The surviving remains of the Haldum casket show a surprising similarity to the Bamberg casket. There is, however, nothing to indicate that the casket from Haldum was as magnificently decorated, but the now completely vanished oak wood casket may possibly have been decorated with both carvings and paintings. Furthermore, the casket originally had edge fittings greater than 3 mm in width which, in themselves, would also have constituted considerable ornamentation. This fact became evident from construction of the replica (fig. 14). The latter also confirmed the reconstruction of the Haldum casket and its complicated closing mechanism.The Haldum find shows that the Bamberg casket, with its special construction, is not unique, and two further finds kept at Danmark’s National Museum indicate that caskets of this type were perhaps more widespread than previously assumed. One is a cruciform fitting of gilt bronze (fig. 15). The four transepts end in stylised animal heads, and at the centre is a hemispherical raised area. At the centre of the lid of the Bamberg casket there is a cruciform fitting also with animal heads at the ends of the transepts, and in the middle sits a hemispherical rock crystal (fig. 16). The similarity to the former fitting is striking, and it seems likely that the artefact represents a lid fitting for a casket of Bamberg type. The other artefact is a cruciform fitting of sheet bronze with open-work sections between the limbs of the cross and a circular hole at its centre (fig. 17). The fitting is part-finished and of the same type as the first mentioned, but a somewhat different variant. The two fittings were found in an old ford across Halleby Å in Western Zealand near the rich Viking Age settlement at Tissø. They were recovered together with the remains of a tool chest.The grave in which the Haldum casket was found is presumed to be that of a woman because beads and small locked caskets are typical woman’s equipment in Viking Age graves. However, such grave goods have also been found in a few cases in men’s graves. The whetstone gives no indication of the sex of the deceased because this type of artefact was commonly included as grave goods in both men’s and women’s graves. The great similarity of Haldum casket to the Bamberg casket dates the grave to the second half of the 10th century.The style of the Bamberg casket indicates that it was produced in Denmark or Southern Scandinavia. Recently, however, attention has been drawn to the fact that the Mammen style also appears over a wider area. Finds from areas where the Vikings settled outside Scandinavia– from The British Isles to Russia – indicate that craft work in the Mammen style could also have been produced there. The finding of the Haldum casket does, however, add weight to the conclusion that the Bamberg casket was produced in Denmark. This is also the case for the two fittings from Halleby Å if the interpretation presented here is correct. However, whether boxes of this type were produced in one particular place or are the work of one or more travelling craftsmen remains to be ascertained.Jens JeppesenMarianne SchwartzMoesgård Museum

The purpose of this study was to test whether the severity of the cranial phenotype in Muenke syndrome infants with unicoronal synostosis is greater than in infants with nonsyndromic unicoronal synostosis. A total of 23 infants were included in the study. All infants included in the study had a computed tomography (CT)-verified synostosis of the coronal suture. The patients were either placed into the "Muenke" group (n=11) or the "non-Muenke" control group (n=12) on the basis of a test for the P250R mutation in the FGFR3 gene. On the basis of CT scans, a three-dimensional surface model corresponding to bone was created for each individual. The sutures were inspected for synostosis, and the degree of synostosis was assessed. Increased digital markings were recorded for both groups. Craniofacial morphology was assessed quantitatively using bony landmarks and recording of the midsagittal surface of the calvaria, cranial base, and maxillary complex. Increased digital markings were more severe posteriorly in Muenke patients than in non-Muenke patients. The Muenke patients with unilateral coronal synostosis showed a somewhat more severe asymmetry in the anterior part of the skull than the non-Muenke patients. The study indicates differences with regard to severity of increased digital markings and craniofacial asymmetry between the infants with Muenke syndrome and the infants with nonsyndromic unilateral coronal synostosis.

Carbohydrate-deficient glycoprotein syndrome type 1A (CDGS1A) is an inherited disorder with multisystemic abnormalities resulting from failure to generate sufficient lipid-linked oligosaccharide precursor or to transfer the sugar chain to many glycoproteins. Cultured fibroblasts from these patients have reduced incorporation of mannose into glycoproteins which can be corrected by adding D-mannose to the culture medium. Providing dietary mannose to elevate mannose concentrations in vivo therefore might remedy some of the underglycosylation in the patients. Five children with CDGS1A aged 15 months to 14 y completed a protocol of enteral supplementation with D-mannose 100 mg/kg every 3 h for 9 d. The mean S-mannose level increased from 32 microM (range 22-42 microM) to a trough value of 72 microM (range 39-103 microM). No serious side effects were observed. Surprisingly, the mean serum concentration of four glycoproteins (transferrin, alpha1-antitrypsin, antithrombin, and thyroxine-binding globulin) tended to decrease, and the mean serum concentration of carbohydrate-deficient transferrin (CDT) increased. Furthermore, the initially present abnormal isoforms of these glycoproteins and of protein C became more prominent and/or additional abnormal isoforms appeared. This short-term trial does not support a benefit of mannose to the deficient glycosylation of CDGS1A patients.

Rett syndrome is a largely sporadic, X-linked neurological disorder with a characteristic phenotype, but which exhibits substantial phenotypic variability. This variability has been partly attributed to an effect of X chromosome inactivation (XCI). There have been conflicting reports regarding incidence of skewed X inactivation in Rett syndrome. In rare familial cases of Rett syndrome, favourably skewed X inactivation has been found in phenotypically normal carrier mothers. We have investigated the X inactivation pattern in DNA from blood and buccal cells of sporadic Rett patients (n=96) and their mothers (n=84). The mean degree of skewing in blood was higher in patients (70.7%) than controls (64.9%). Unexpectedly, the mothers of these patients also had a higher mean degree of skewing in blood (70.8%) than controls. In accordance with these findings, the frequency of skewed (XCIor =80%) X inactivation in blood was also higher in both patients (25%) and mothers (30%) than in controls (11%). To test whether the Rett patients with skewed X inactivation were daughters of skewed mothers, 49 mother-daughter pairs were analysed. Of 14 patients with skewed X inactivation, only three had a mother with skewed X inactivation. Among patients, mildly affected cases were shown to be more skewed than more severely affected cases, and there was a trend towards preferential inactivation of the paternally inherited X chromosome in skewed cases. These findings, particularly the greater degree of X inactivation skewing in Rett syndrome patients, are of potential significance in the analysis of genotype-phenotype correlations in Rett syndrome.

Objectives The prevalence of limb girdle muscular dystrophy type 2I (LGMD2I) in northern Europe is unknown. We investigated this and the genotype–phenotype relation in LGMD2I. Methods Prospective clinical and molecular screening of 118 Danish patients registered with LGMD was performed to divide patients into LGMD subtypes. Results One hundred three patients fulfilled the clinical criteria for LGMD2. Thirty-eight had LGMD2I (27 homozygous, 11 compound heterozygous for 826C>A), 23 had sarcoglycanopathy, 2 dysferlinopathy, 12 calpainopathy, and 4 Becker muscular dystrophy. The 24 patients with no molecular diagnosis did not harbor fukutin-related protein gene (FKRP) mutations. A clear clinical delineation was found between patients homozygous and compound heterozygous for the 826C>A mutation. Homozygous patients had later debut, milder clinical progression, and less muscle weakness compared with compound heterozygous patients, who were all wheelchair bound by their mid-20s. Impaired cardiac pump function was found in both groups. Interpretation This study reports a different distribution of LGMD subtypes in Denmark than seen in other geographic regions, with a threefold to fourfold higher prevalence of LGMD2I than elsewhere. The findings support a clear clinical delineation between patients homozygous and compound heterozygous for the 826C>A mutation in FKRP. The findings suggest that, in the studied region, screening for the 826C>A mutation will identify all persons with LGMD2I. Ann Neurol 2006;59:808–815

The diagnosis of hereditary non-polyposis colorectal cancer (HNPCC) is often confirmed by a mutation in one of several mismatch-repair genes, in particular MLH1, MSH2 and MSH6. Presymptomatic diagnosis requires the identification of a mutation causing the disease. Three different deletions of a single amino acid codon have previously been published as assumed pathogenic. The objective of this study was to determine if an MSH2 3 base pair in-frame deletion (N596del) could be used in presymptomatic screening of at-risk individuals. We report on five HNPCC families with the N596del mutation, identified after mutation screening of MSH2 and MLH1. All patients in the families were haplotyped using markers flanking the MSH2 gene. The haplotypes revealed that the five families with high probability descended from only two founders. The N596del segregated with the HNPCC phenotype with lod scores of 3.2 and 2.0 at the recombination fraction of 0.0 in the two founder families. Sequencing of MSH2 and MLH1 did not reveal other pathogenic mutations, and N596del was not identified in 50 healthy controls. The mutation has previously been found expressed in mRNA, and is located in a conserved domain. The results support the hypothesis that N596del is the disease causing mutation and not a clinically silent variation. On this basis, the application of the MSH2 N596del mutation, in presymptomatic screening of HNPCC families, is recommended.

Patients with the myopathic form of carnitine palmitoyltransferase II (CPT II) deficiency typically experience muscle pain, cramps, and myoglobinuria during prolonged exercise. It has been suggested that carriers of CPT2 gene mutations also may have milder clinical symptoms, but fatty acid oxidation (FAO) has never been investigated in vivo in this group. We studied fuel utilization by indirect calorimetry and stable isotope methodology in four patients with CPT II deficiency, three subjects who carried one CPT2 gene mutation, and five healthy control subjects. Cycle exercise at a constant workload of 50% of maximal oxygen uptake capacity was used to facilitate FAO. We found that in vivo oxidation of long-chain fatty acids was normal at rest but severely impaired during prolonged, low-intensity exercise in patients with CPT II deficiency, and that two of the single CPT2 gene mutation carriers, who displayed symptoms of CPT II deficiency, had an FAO comparable with the patients. These results indicate that residual CPT II activity is sufficient to maintain long-chain FAO at rest in CPT II deficiency but not to increase FAO during exercise. The findings also suggest that single CPT2 gene mutations may exert a dominant-negative effect on the tetrameric CPT II protein.

Mutation detection in the DMD gene defective in Duchenne (DMD) and Becker muscular dystrophies (BMD) is complicated by the presence of 79 exons. The majority of recognized mutations are, however, copy number changes of individual exons, which traditionally have been identified by three common multiplex polymerase chain reaction (PCR) assays and/or Southern blotting. Here we report the use of the newly developed quantitative assay multiplex ligation-dependent probe amplification (MLPA) to determine the copy number of each of the 79 DMD exons in 182 males and 14 carrier females referred to our diagnostic facility on the clinical suspicion of DMD or BMD. The MLPA method confirmed all previously recognized mutations and identified an additional 28, including four point mutations. Also, the assay reliably identified 7 carrier females, which are usually not easily recognized. In our hands the method is highly reproducible, easy to handle, and has increased our mutation pick-up rate by a total of 33)%.

Mutations in the CLCN1 gene, encoding a muscle-specific chloride channel, can cause either recessive or dominant myotonia congenita (MC). The recessive form, Becker's myotonia, is believed to be caused by two loss-of-function mutations, whereas the dominant form, Thomsen's myotonia, is assumed to be a consequence of a dominant-negative effect. However, a subset of CLCN1 mutations can cause both recessive and dominant MC. We have identified two recessive and two dominant MC families segregating the common R894X mutation. Real-time quantitative RT-PCR did not reveal any obvious association between the total CLCN1 mRNA level in muscle and the mode of inheritance, but the dominant family with the most severe phenotype expressed twice the expected amount of the R894X mRNA allele. Variation in allelic expression has not previously been described for CLCN1, and our finding suggests that allelic variation may be an important modifier of disease progression in myotonia congenita.

Voltage-gated ClC-1 chloride channels encoded by the CLCN1 gene have a major role in setting the membrane potential in skeletal muscle. More than 60 CLCN1 mutations have been associated with myotonia congenita. These mutations are traditionally classified as recessive (Becker's disease) or dominant (Thomsen's disease). In this study, we have electrophysiologically characterized two new dominant ClC-1 mutations, thereby elucidating the observed phenotype in patients. The two ClC-1 mutants M128V and E193K were identified, and the DNA was isolated from patients and subsequently expressed in Xenopus laevis oocytes for electrophysiological characterization. Both ClC-1 mutants, M128V and E193K, showed a large rightward shift in the current–voltage relationship. In addition, the activation kinetics were slowed in the ClC-1 M128V mutant, as compared to the wild-type ClC-1. Interestingly, ClC-1 E193K revealed a change in reversal potential compared to wild-type channels. This finding supports the notion that the E193 amino acid is an important determinant in the selectivity filter of the human ClC-1 channel. The electrophysiological behavior of both mutants demonstrates a severe reduction in ClC-1 channel conductance under physiologically relevant membrane potentials. These studies thereby explain the molecular background for the observed myotonia in patients. Muscle Nerve 28: 722–732, 2003

The purpose of this study was to investigate the correlation between the level of mutated mitochondrial DNA in muscle and oxidative capacity in 24 patients with mitochondrial myopathy (MM). Maximal oxygen uptake (VO(2max)), workload (W(max)), and venous plasma lactate levels were measured during an incremental cycle test to exhaustion in 17 patients with point mutations of mtDNA and in seven with single, large-scale deletions of mtDNA (chronic progressive external ophthalmoplegia [CPEO]). Results were compared with those in 25 healthy matched subjects. The mutation load in MM patients was 67 +/- 5% (range, 29 - 99%). VO(2max) and W(max) correlated with percentage of heteroplasmy (r0.82; p0.005) and were lower in patients versus healthy subjects (p0.000005). Exercise-induced peak increases in heart rate, ventilation, and resting plasma lactate levels correlated with muscle mutation load (r0.71; p0.005). Exercise-induced increases in plasma lactate correlated with muscle mutation load in CPEO patients (r = 0.95; p0.005). Impaired oxidative capacity and ragged red muscle fibers were found in CPEO and 3243A--G patients with mutation loads as low as 45 and 57%, respectively. The study indicates that oxidative capacity correlates directly with skeletal muscle mutation load in MM patients, and that the mutation threshold level for impaired oxidative metabolism in MM patients is lower than found in in vitro studies.

Decrement of the compound muscle action potential (CMAP) during 10-HZ repetitive nerve stimulation is thought to be an unusual finding in dominant myotonia congenita, and has not previously been reported in patients with the genetically verified disorder. It was the purpose of the present study to elucidate the relation between decrement and CLCN1 mutation type in myotonia congenita. Decrement and genotypes were studied in eight Danish families with myotonia congenita. Six patients with the known dominant mutation P480L had decrements of 30–84%. Patients heterozygous for the R894X mutation had decrements of 20–47%. Three novel CLCN1 mutations (two dominant and one recessive) were found segregating with the Thomsen/Becker phenotypes. In families with the novel dominant mutations M128V and E193K, decrement was absent in all family members tested. In conclusion, CMAP decrement may be pronounced in dominant myotonia congenita, and the presence of decrement is related to mutation type. Muscle Nerve 27:449–455, 2003

We recently showed that mutations in the CNGA3 gene encoding the alpha -subunit of the cone photoreceptor cGMP-gated channel cause autosomal recessive complete achromatopsia linked to chromosome 2q11. We now report the results of a first comprehensive screening for CNGA3 mutations in a cohort of 258 additional independent families with hereditary cone photoreceptor disorders. CNGA3 mutations were detected not only in patients with the complete form of achromatopsia but also in incomplete achromats with residual cone photoreceptor function and (rarely) in patients with evidence for severe progressive cone dystrophy. In total, mutations were identified in 53 independent families comprising 38 new CNGA3 mutations, in addition to the 8 mutations reported elsewhere. Apparently, both mutant alleles were identified in 47 families, including 16 families with presumed homozygous mutations and 31 families with two heterozygous mutations. Single heterozygous mutations were identified in six additional families. The majority of all known CNGA3 mutations (39/46) are amino acid substitutions compared with only four stop-codon mutations, two 1-bp insertions and one 3-bp in-frame deletion. The missense mutations mostly affect amino acids conserved among the members of the cyclic nucleotide gated (CNG) channel family and cluster at the cytoplasmic face of transmembrane domains (TM) S1 and S2, in TM S4, and in the cGMP-binding domain. Several mutations were identified recurrently (e.g., R277C, R283W, R436W, and F547L). These four mutations account for 41.8% of all detected mutant CNGA3 alleles. Haplotype analysis suggests that the R436W and F547L mutant alleles have multiple origins, whereas we found evidence that the R283W alleles, which are particularly frequent among patients from Scandinavia and northern Italy, have a common origin.

The Wiskott-Aldrich syndrome protein (WASP; encoded by the gene WAS) and its homologs are important regulators of the actin cytoskeleton, mediating communication between Rho-family GTPases and the actin nucleation/crosslinking factor, the Arp2/3 complex. Many WAS mutations impair cytoskeletal control in hematopoietic tissues, resulting in functional and developmental defects that define the X-linked Wiskott-Aldrich syndrome (WAS) and the related X-linked thrombocytopenia (XLT). These diseases seem to result from reduced WASP signaling, often through decreased transcription or translation of the gene. Here we describe a new disease, X-linked severe congenital neutropenia (XLN), caused by a novel L270P mutation in the region of WAS encoding the conserved GTPase binding domain (GBD). In vitro, the mutant protein is constitutively activated through disruption of an autoinhibitory domain in the wild-type protein, indicating that loss of WASP autoinhibition is a key event in XLN. Our findings highlight the importance of precise regulation of WASP in hematopoietic development and function, as impairment versus enhancement of its activity give rise to distinct spectra of cellular defects and clinical phenotypes.

Mitochondrial cytochrome b mutations have been reported to have a homogenous phenotype of pure exercise intolerance. We describe a novel mutation in the cytochrome b gene of mitochondrial DNA (A15579G) associated with a selective decrease of muscle complex III activity in a patient who, besides severe exercise intolerance, also has multisystem manifestations (deafness, mental retardation, retinitis pigmentosa, cataract, growth retardation, epilepsy). The point mutation is heteroplasmic in muscle (88%) and leukocytes (15%), and changes a highly conserved tyrosine to cysteine at amino acid position 278.

Propionyl-CoA carboxylase (PCC) catalyzes the biotin-dependent carboxylation of propionyl-CoA to d-methylmalonyl-CoA in the mitochondrial matrix. Human PCC is a dodecamer composed of pairs of nonidentical alpha and beta subunits encoded by PCCA and PCCB genes, respectively. Deficiency of PCC results in propionic acidemia (PA), a metabolic disorder characterized by severe metabolic ketoacidosis, vomiting, lethargy, and hypotonia. To date, almost 60 mutations have been reported in both genes. Exon 15 of the beta subunit is one of the two sites where a number of mutations have been identified in PA patients. In the primary betaPCC sequence, these mutations lead to three substitutions (R512C, L519P, and N536D), three truncations (R499X, R514X, and W531X), and one insertion (A51_R514insP). We expressed these mutant proteins in Escherichia coli in which the GroESL complex was overexpressed. The only mutation that does not impact the stability of mutant betaPCC in bacteria is W531X. The remaining mutations lead to either complete (L519P, N536D) or partial (R499X, R512C, A513_R514insP, and R514X) degradation of the mutant subunits. Size-exclusion chromatography revealed that R512C and W531X do not affect the assembly of alphaPCC and betaPCC to active oligomers. Specific activities for these mutant proteins, however, were only 3.9 and 10% of the wild type, respectively. Taken together, the carboxyl-terminal portion of 40 amino acid residues of the beta subunit affects the stability and the assembly of the alpha and beta subunits as well as the carboxylation of propionyl-CoA.

Mutations in the androgen receptor gene are considered as incompatible with preservation of fertility and have been suggested as a cause of male infertility.Two adult brothers, referred because of gynecomastia and hormonal levels in serum indicating androgen insensitivity (high sex hormone-binding globulin, and LH levels, despite extremely high testosterone concentration), turned out to be relatives to a third young man, referred independently of the two others and exhibiting identical clinical and hormonal stigmata. In all three men, we found a C→A substitution at position 2470 (exon 7) in the androgen receptor gene, leading to a Gln824Lys mutation in the ligand-binding domain of the receptor. Exploring the family history revealed that their grandfathers, on their mothers’ side, were brothers; and the Gln824Lys mutation was also found in the one of them who was still alive.Binding studies with the mutant receptor in transfected COS-7 cells, with mibolerone as ligand, exhibited equal Kd (0.7 vs. 1.0 nmol/L), IC50 (0.8 vs. 1.1 nmol/L), and maximum binding (7.1 vs. 8.9 fmol/106 cells), as compared with the wild-type (WT) receptor. In a chloramphenicol acetyl transferase trans-activation assay, the activity of the mutant receptor was identical to that of the WT, when the synthetic androgen R1881 was used as a ligand; but with dihydrotestosterone, in concentrations up to 10 nmol/L, the activity of Gln824Lys mutated receptor was 10–62% of the WT variant.Thus, Gln824Lys mutation was found, both in vivo and in vitro, to cause slight impairment of receptor function but was compatible with preservation of male fertility. The patients inherited the mutation from their grandfathers through their mothers, and one of the young men possessing the mutation has fathered a daughter.

Mannose-binding lectin (MBL) is a key factor in innate immunity, and lung infections are the leading cause of morbidity and mortality in cystic fibrosis (CF). Accordingly, we investigated whether MBL variant alleles, which are associated with recurrent infections, might be risk factors for CF patients. In 149 CF patients, different MBL genotypes were compared with respect to lung function, microbiology, and survival to end-stage CF (death or lung transplantation). The lung function was significantly reduced in carriers of MBL variant alleles when compared with normal homozygotes. The negative impact of variant alleles on lung function was especially confined to patients with chronic Pseudomonas aeruginosa infection. Burkholderia cepacia infection was significantly more frequent in carriers of variant alleles than in homozygotes. The risk of end-stage CF among carriers of variant alleles increased 3-fold, and the survival time decreased over a 10-year follow-up period. Moreover, by using a modified life table analysis, we estimated that the predicted age of survival was reduced by 8 years in variant allele carriers when compared with normal homozygotes. Presence of MBL variant alleles is therefore associated with poor prognosis and early death in patients with CF. J. Clin. Invest. 104:431-437 (1999).

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is caused by mutations in the gene CYP21 encoding the enzyme steroid 21-hydroxylase. In addition to deletions, approximately 20 different point mutations have been reported, and still novel mutations are detected. This makes genetic diagnosis as well as carrier detection of 21-hydroxylase deficiency a complicated matter. We developed a simple nonradioactive assay based on the polymerase chain reaction (PCR) in combination with denaturing gradient gel electrophoresis (DGGE) to screen for mutations in the CYP21 gene. DGGE allows a fast scanning of PCR-amplified segments of genes for the presence or absence of any single base pair alterations. We have performed this technique on the coding sequence and intron-exon junctions of CYP21. Our results emphasize that this procedure constitutes a fast and reliable approach when performing diagnosis of 21-hydroxylase deficiency. Hum Mutat 13:385–389, 1999. © 1999 Wiley-Liss, Inc.

We have searched for the delta F508 mutation in 77 Norwegian cystic fibrosis patients. Of the 154 chromosomes tested, 93 (60%) carried the delta F508 mutation. Haplotypes at the D7S23 locus (KM19 and XV2C markers) were determined. Of 81 chromosomes with the F508 mutation, the B haplotype was found on 77. We found three patients with the G551D and one patient with the R553X mutation in exon 11 of the CFTR locus.

Thiamine-responsive megaloblastic anaemia (TRMA) is a rare autosomal recessive condition, characterized by megaloblastic anaemia, non-autoimmune diabetes mellitus, and sensorineural hearing loss. We describe three infants with TRMA from two consanguineous Pakistani families, who were not known to be related but originated from the same area in Pakistan. All children were homozygous, and the parents were heterozygous for a c.196G>T mutation in the SLC19A2 gene on chromosome 1q23.3, which encodes a high-affinity thiamine transporter. The result is an abnormal thiamine transportation and vitamin deficiency in the cells. Thiamine in high doses (100-200 mg/d) reversed the anaemia in all our patients. Two patients discontinued insulin treatment successfully after a short period, while the third patient had to continue with insulin. The hearing loss persisted in all three children. The diagnosis of TRMA should be suspected in patients with syndromic diabetes including hearing loss and anaemia, even if the latter is only very mild and, particularly, in the case of consanguinity.

The chemokine receptor CCR5 is encoded by the CMKBR5 gene located on the p21.3 region of human chromosome 3, and constitutes the major co-receptor for the macrophage-tropic strains of HIV-1. A mutant allele of the CCR5 gene, Delta ccr5 , was shown to provide to homozygotes with a strong resistance against infection by HIV. The frequency of the Delta ccr5 allele was investigated in 18 European populations. A North to South gradient was found, with the highest allele frequencies in Finnish and Mordvinian populations (16%), and the lowest in Sardinia (4%). Highly polymorphic microsatellites (IRI3.1, D3S4579 and IRI3.2, D3S4580 ) located respectively 11 kb upstream and 68 kb downstream of the CCR5 gene deletion were used to determine the haplotype of the chromosomes carrying the Delta ccr5 variant. A strong linkage disequilibrium was found between Delta ccr5 and specific alleles of the IRI3.1 and IRI3.2 microsatellites: >95% of the Delta ccr5 chromosomes carried the IRI3.1-0 allele, while 88% carried the IRI3.2-0 allele. These alleles were found respectively in only 2 or 1.5% of the chromosomes carrying a wild-type CCR5 gene. From these data, it was inferred that most, if not all Delta ccr5 alleles originate from a single mutation event, and that this mutation event probably took place a few thousand years ago in Northeastern Europe. The high frequency of the Delta ccr5 allele in Caucasian populations cannot be explained easily by random genetic drift, suggesting that a selection advantage is or has been associated with homo- or heterozygous carriers of the Delta ccr5 allele.

Glutaric acidemia type I (GA1) is caused by mutations in the gene encoding the enzyme glutaryl-CoA dehydrogenase (GCD). Sixty-three pathogenic mutations identified by several laboratories are presented, 30 of them for the first time, together with data on expression in Escherichia coli and relationship to the clinical and biochemical phenotype. In brief, many GCD mutations cause GA1, but none is common. There is little if any relationship between genotype and clinical phenotype, but some mutations, even when heterozygous, seem especially common in patients with normal or only minimally elevated urine glutaric acid.

Choroideremia (CHM) is an X-linked recessive eye disease that results from mutations involving the Rab escort protein-1 (REP-1) gene. In 18 patients deletions of different sizes have been found. Two females suffering from CHM were reported to have translocations that disrupt the REP-1 gene. In 22 patients, small mutations have been identified. Interestingly, these are all nonsense, frameshift or splice-site mutations; with one possible exception, missense mutations have not been found. This comprises all the known mutations in the disease. Hum Mutat 9:110–117, 1997. © 1997 Wiley-Liss, Inc.