Mutations in the gene-encoding SERCA1, the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+ ATPase, are associated with Brody disease.

Brody disease is a rare inherited disorder of skeletal muscle function. Symptoms include exercise-induced impairment of skeletal muscle relaxation, stiffness and cramps. Ca2+ uptake and Ca2+ ATPase activities are reduced in the sarcoplasmic reticulum, leading to the prediction that Brody disease results from defects in the ATP2A1 gene on chromosome 16p12.1-12.2, encoding SERCA1, the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+ ATPase. A recent search, however, did not reveal any mutations in the ATP2A1 gene in three Brody patients. We have now associated Brody disease with the autosomal recessive inheritance of three ATP2A1 mutations in two families, suggesting that the disease is genetically heterogeneous. One mutation occurs at the splice donor site of intron 3, while the other two mutations lead to premature stop codons, truncating SERCA1, deleting essential functional domains and raising the intriguing question: how have these Brody patients partially compensated for the functional knockout of a gene product believed to be essential for fast-twitch skeletal muscle relaxation?

Mutations in SDHD, a mitochondrial complex II gene, in hereditary paraganglioma.

Hereditary paraganglioma (PGL) is characterized by the development of benign, vascularized tumors in the head and neck. The most common tumor site is the carotid body (CB), a chemoreceptive organ that senses oxygen levels in the blood. Analysis of families carrying the PGL1 gene, described here, revealed germ line mutations in the SDHD gene on chromosome 11q23. SDHD encodes a mitochondrial respiratory chain protein-the small subunit of cytochrome b in succinate-ubiquinone oxidoreductase (cybS). In contrast to expectations based on the inheritance pattern of PGL, the SDHD gene showed no evidence of imprinting. These findings indicate that mitochondria play an important role in the pathogenesis of certain tumors and that cybS plays a role in normal CB physiology.

Increased prevalence of catecholamine excess and phaeochromocytomas in a well-defined Dutch population with SDHD-linked head and neck paragangliomas.

OBJECTIVE: The aim of this study was to identify the prevalence of catecholamine excess and phaeochromocytomas in a well-defined population of people with hereditary head and neck paragangliomas. METHODS: We studied in a prospective follow-up protocol all consecutive patients referred to the Department of Endocrinology, Leiden University Medical Center, Leiden, The Netherlands with documented head and neck paragangliomas and either a positive family history for paragangliomas or a proven SDHD gene mutation. Initial analysis included medical history, physical examination and the measurement of excretion of catecholamines in two 24-h urine collections. In the case of documented catecholamine excess iodinated meta-iodobenzylguanidine (123I-MIBG) scintigraphy and magnetic resonance imaging were done. RESULTS: Between 1988 and 2003, 40 consecutive patients (20 male and 20 female) with documented head and neck paragangliomas were screened. Biochemical screening revealed urinary catecholamine excess in 15 patients (37.5%). In nine of these 15 patients a lesion was found by 123I-MIBG scintigraphy. Exact localization by magnetic resonance imaging revealed phaeochromocytomas in seven of the 15 patients. One of the nine patients had an extra-adrenal paraganglioma. Histopathological examination in a subset of tumors displayed loss of heterozygosity of the wild-type SDHD allele in all cases. CONCLUSIONS: The prevalence of catecholamine excess (37.5%) and phaeochromocytomas (20.0%) is high in patients with familial head and neck paragangliomas. Therefore, patients with hereditary head and neck paragangliomas require lifelong follow up by biochemical testing for catecholamine excess.

Phenotypic dichotomy in mitochondrial complex II genetic disorders.

This review presents our current knowledge on the genetic and phenotypic aspects of mitochondrial complex II gene defects. The mutations of the complex II subunits cause two strikingly different group of disorders, revealing a phenotypic dichotomy. Genetic disorders of the mitochondrial respiratory chain are often characterized by hypotonia, growth retardation, cardiomyopathy, myopathy, neuropathy, organ failure, and metabolic derangement. These disorders are transmitted through maternal lineage if the defective gene is located in the mitochondrial genome or may follow a Mendelian pattern if it is in the nucleus. Mitochondrial complex II (succinate:ubiquinone oxidoreductase) is the smallest complex in the respiratory chain and is composed of four subunits encoded by nuclear genes SDHA, SDHB, SDHC, and SDHD. Complex II oxidizes succinate to fumarate in the Krebs cycle and is involved in the mitochondrial electron transport chain. SDHA and SDHB encode the flavoprotein and iron-sulfur proteins, respectively, and SDHC and SDHD encode the two hydrophobic membrane-spanning subunits. While mutations in SDHA display a phenotype resembling other mitochondrial and Krebs cycle gene defects, those in SDHB, SDHC and SDHD cause hereditary paraganglioma. Paraganglioma is characterized by slow-growing vascular tumors of the paraganglionic tissue (i.e., adrenal and extra-adrenal paragangliomas, including those in the head and neck, mediastinum, abdomen, and pheochromocytomas). Paraganglioma caused by SDHD mutations occurs exclusively after paternal transmission, suggesting that genomic imprinting influences gene expression. Association of a mitochondrial gene defect with tumorigenesis expands the phenotypic spectrum of mitochondrial diseases and adds genomic imprinting as a new transmission mode in mitochondrial genetics. The phenotypic features of complex II gene mutations suggest that whereas the catalytic subunit SDHA mutations may compromise the Krebs cycle, those in other structural subunits may affect oxygen sensing and signaling.

[From gene to disease; from CLN1, CLN2 and CLN3 to neuronal ceroid lipofuscinosis]. / Van gen naar ziekte; van Clni, CLN2 en CLN3 naar neuronale ceroidlipofuscinose.

The neuronal ceroid lipofuscinoses (NCL) are worldwide the most common lysosomal storage disorders of childhood. Clinical features often include progressive visual impairment, seizures, psychomotor deterioration, dementia, and premature death. Most NCL cases are caused by mutations in the CLN1, CLN2 and CLN3 genes, which play an essential role in lysosomal protein degradation. Laboratory diagnostics for a patient suspected of NCL should start with enzyme analysis in the case of INCL and LINCL and investigation of lymphocyte vacuolisation for JNCL. Diagnosis at the protein level is not available for JNCL, but CLN3 mutation analysis is possible. The carrier status of healthy relatives in families with known mutations in either CLN1, CLN2, CLN3 or CLN6 can be determined with certainty by mutation analysis.

A high-resolution integrated map spanning the SDHD gene at 11q23: a 1.1-Mb BAC contig, a partial transcript map and 15 new repeat polymorphisms in a tumour-suppressor region.

Chromosomal region 11q22-q23 is a frequent target for deletion during the development of many solid tumour types, including breast, ovary, cervix, stomach, bladder carcinomas and melanoma. One of the most commonly deleted subregions contains the SDHD gene, which encodes the small subunit of cytochrome b (cybS) in mitochondrial complex II (succinate-ubiquinone oxidoreductase). Germline mutations in SDHD cause hereditary paraganglioma type 1 (PGL1), and suggest a tumour suppressor role for cybS. We present a high-resolution physical map spanning SDHD, covered by 19 YACs and 20 BACs. An approximate 1.1-Mb gene-rich region around SDHD is spanned by a complete BAC contig. Twenty-six new STSs are developed from the BAC clone ends. In addition to the discovery and characterisation of 15 new simple tandem repeat polymorphisms, we provide integrated positional information for 33 ESTs and known genes, including KIAA1391, POU2AF1 (OBF1), PPP2R1B, CRYAB, HSPB2, DLAT, IL-18, PTPS, KIAA0781 and KAIA4591, which is mapped by NotI site cloning. We describe full-length transcript sequence for PPP2R1B, encoding the protein phosphatase 2A regulatory subunit A beta isoform. We also discover a processed pseudogene for USA-CYP, a cyclophilin associated with U4/U6 snRPNs, and a novel gene, DDP2, encoding a mitochondrial protein similar to the X-linked deafness-dystonia protein, which is juxtaposed 5'-to-5' to SDHD. This map will help assess this gene-rich region in PGL and in other common tumours.

Late onset juvenile neuronal ceroid-lipofuscinosis with granular osmiophilic deposits (GROD).

The juvenile-onset subtype of the neuronal ceroid lipofuscinoses (JNCL) is well known [Hofman, ISBN90-71534-19-7 1990] and ultrastructurally characterized by fingerprints and/or curvilinear bodies in many cell types. Linkage studies indicated a most likely location for CLN3, the gene involved in JNCL, in the interval between loci D16S297 and D16S57, within close proximity of the loci D16S298 and D16S299 [Mitchison et al., Genomics 22:465-468, 1993]. We present two sibs with a late onset progressive disease of mental deterioration, progressive macular degeneration, motor disturbances, and epilepsy. Histological symptoms of neuronal ceroid lipofuscinosis and ultrastructural granular osmiophilic deposits (GROD) in lymphocytes and neurons are found. Individual haplotypes at polymorphic marker loci on chromosome 16 were constructed to determine whether JNCL with GROD is linked to the CLN3 locus.

Physical map of the region containing the gene for Batten disease (CLN3).

CLN3 has been mapped genetically to 16p12, to the interval between D16S288 and D16S383, a sex-averaged genetic distance of 2.1 cM. Analysis of disease haplotypes for four microsatellite markers in this interval, D16S288, D16S299, D16S298, and SPN, has shown significant allelic association between one allele at each of these loci and CLN3. All four of the associated markers were used as nucleation sites in the isolation of genomic clones (YACs). A contig was assembled which contains 3 of the 4 associated markers and which confirmed the relative order of these markers. Marker D16S272 has been located on the physical map between D16S288 and D16S299. Restriction mapping has demonstrated the location of possible CpG islands. One gene, STP, has been localised on the YAC contig proximal to D16S298 and is therefore a candidate for CLN3. Other genes, including IL4R, SGLT2, and UQCRC2, have been excluded from this region.

Isolation of genes from the Batten candidate region using exon amplification. Batten Disease Consortium.

In order to identify genes originating from the Batten disease candidate region, we have used the technique of exon amplification to identify transcribed sequences. This procedure produces trapped exon clones, which can represent single exons or multiple exons spliced together and is an efficient method for obtaining probes for physical mapping and for screening cDNA libraries. The source of DNA for these experiments was a collection of chromosome 16 cosmid contigs isolated by the direct subcloning of region-specific yeast artificial chromosomes (YACs) and hybridization of inter-alu PCR products from these YACs to the flow-sorted Los Alamos chromosome 16 cosmid library. We are now using the resulting exon probes to screen retina and brain cDNA libraries for candidate JNCL genes.

Application of chromosome 16 markers in the differential diagnosis of neuronal ceroid-lipofuscinosis.

Accurate diagnosis of neuronal ceroid lipofuscinosis (NCL) is important for a correct prognosis of the disease and for genetic counseling. Up to now, no direct diagnostic test has been available for NCL. The clinical diagnosis is made on the basis of symptoms, neurophysiological, neuroradiological, and specific lipopigment pattern data. Recent advances in the genetics of NCL have enabled us to use polymorphic DNA markers linked to the CLN1 and CLN3 loci as a tool in the differential diagnosis of NCL. We have applied genetic analysis with polymorphic DNA markers flanking the CLN3 gene on chromosome 16 to two consanguineous families in which NCL occurs. In the first family, which is of Turkish extraction, two patients suffering from a protracted form of juvenile NCL previously had been diagnosed with juvenile NCL. Haplotypes from this family indicate that the patients and their healthy sibling are haplo-identical, suggesting that this protracted form of juvenile NCL is not linked to the CLN3 locus. In the second family, which is of Moroccan origin, one patient suffers from the early juvenile variant of NCL (Lake-Cavanagh). In this family, the patient and one of the healthy siblings have identical haplotypes, excluding linkage of early juvenile NCL to the CLN3 locus on 16p12.1-11.2. Therefore, these cases from different populations demonstrate that haplotype analysis can be used as an additional method to exclude the diagnosis of juvenile NCL.

Refined localization of the Batten disease gene (CLN3) by haplotype and linkage disequilibrium mapping to D16S288-D16S383 and exclusion from this region of a variant form of Batten disease with granular osmiophilic deposits.

Haplotype analysis in a collaborative collection of 143 families with juvenile-onset neuronal ceroid lipofuscinosis (JNCL) or Batten (Spielmeyer-Vogt-Sjögren) disease has permitted refined localization of the disease gene, CLN3, which was assigned to chromosome 16 in 1989. Recombination events in four maternal meioses delimit new flanking genetic markers for CLN3 which localize the gene to the chromosome interval 16p12.1-11.2 between microsatellite markers D16S288 and D16S383. This narrows the position of CLN3 to a region of 2.1 cM, a significant reduction from the previous best interval. Using haplotypes, analysis of the strong linkage disequilibrium that exists between genetic markers within the D16S288-D16S383 interval and CLN3 shows that CLN3 is in closest proximity to loci D16S299 and D16S298. Analysis of markers across the D16S288-D16S383 region in four families with a variant form of JNCL characterized histologically by cytosomal granular osmiophilic deposits (GROD) has excluded linkage of the gene locus to the CLN3 region of chromosome 16, suggesting that JNCL with GROD is not an allelic form of JNCL.

Carrier detection of Batten disease (juvenile neuronal ceroid-lipofuscinosis).

Batten disease, or the juvenile form of neuronal ceroid lipofuscinosis, is an autosomal recessive neurodegenerative disorder manifesting with progressive blindness, seizures, and dementia, leading to an early death. The CLN3 locus which is involved in Batten disease had been localized to chromosome 16p11.2. Linkage disequilibrium has been observed between CLN3 and polymorphic microsatellite markers D16S288, D16S299, and D16S298, making carrier detection and prenatal diagnosis by haplotype analysis possible. For the purpose of carrier detection, haplotypes from Dutch Batten patients and their families were constructed. Most patients share the same D16S298 allele, suggesting the presence of a founder effect in the Dutch population. In a large inbred Dutch family, in which Batten disease occurs with high frequency, haplotype analysis has been carried out with high accuracy for carrier detection.

Caenorhabditis elegans homologues of the CLN3 gene, mutated in juvenile neuronal ceroid lipofuscinosis.

Neuronal ceroid lipofuscinoses (NCLs) are the most common hereditary neurodegenerative disorders of childhood. The first symptom of this heterogeneous group of devastating lysosomal storage diseases is progressive visual failure. The different forms of NCL can be distinguished by age of onset, clinical features and the characteristics of the accumulated materials. The juvenile form, Batten-Spielmeyer-Vogt disease which is caused by mutations in the CLN3 gene, is the most frequent form of the disease in which loss of vision becomes apparent around the age of 5-8 years. The gene was found to encode a novel integral membrane protein localizing to the lysosomes, confirming that the primary defect in NCL is in lysosomal function. The CLN3 protein function is still unknown, and is examined in several model organisms. We are studying the nematode Caenorhabditis elegans, and have identified three CLN3 homologues. In order to investigate the role of the CLN3 protein in C. elegans, Cecln-3 deletion mutants are being isolated from an ethyl methanesulphonate (EMS)-induced deletion mutant library. Examination of these mutants may provide us with information that will help in dissecting the processes in which the CLN3 protein is involved. In this library two mutated C. elegans Cln-3 loci have been identified, of which one mutant, NL748, was isolated. This mutant contains a deletion of the whole gene. The deletion mutant was characterized with regard to life expectancy, and showed no significant differences when compared with wild-type.

New mutations in the neuronal ceroid lipofuscinosis genes.

Thirty-eight mutations and seven polymorphisms have recently been reported in the genes underlying the neuronal ceroid lipofuscinoses (NCLs) including 11 new mutations described here. A total of 114 mutations and 28 polymorphisms have now been described in the five human genes identified which cause NCL. Thirty-eight mutations are recorded for CLN1/PPT; 40 for CLN2/TTP-1, 31 for CLN3, four for CLN5, one for CLN8. Two mutations have been described in animal genes (cln8/mnd, CTSD). All mutations in NCL genes are contained in the NCL Mutation Database (http://www.ucl.ac.uk/NCL).

[From gene to disease; from SDHD, a defect in the respiratory chain, to paragangliomas and pheochromocytomas]. / Van gen naar ziekte; van SDHD, een defect in de ademhalingsketen, naar paragangliomen en feochromocytomen.

Hereditary paragangliomas are rare benign tumours arising from neuroectodermal tissue in the head and neck region. In families with paraganglioma, occasionally adrenal and extra-adrenal pheochromocytomas are found. Paragangliomas, adrenal and extra-adrenal pheochromocytomas may be caused by mutations in the SDHB, SDHC and SDHD genes encoding different subunits of mitochondrial respiratory chain complex II. Most paraganglioma cases in the Netherlands are caused by SDHD mutations. Presymptomatic DNA diagnosis is available for families with paragangliomas caused by SDHD mutations.

Deletion of the Caenorhabditis elegans homologues of the CLN3 gene, involved in human juvenile neuronal ceroid lipofuscinosis, causes a mild progeric phenotype.

The CLN3 gene is involved in juvenile neuronal ceroid lipofuscinosis (JNCL), or Batten-Spielmeyer-Vogt disease, a severe hereditary neurodegenerative lysosomal storage disorder characterized by progressive disease pathology, with loss of vision as the first symptom. Another characteristic of JNCL is the lysosomal accumulation of autofluorescent lipopigments, forming fingerprint storage patterns visible by electron microscopy. The function of the CLN3 protein is still unknown, although the evolutionarily conserved CLN3 protein is being functionally analysed using different experimental models. We have explored the potential of the nematode Caenorhabditis elegans as a model for Batten disease in order to bridge the gap between the unicellular yeast and very complex mouse JNCL models. C. elegans has three genes homologous to CLN3, for each of which deletion mutants were isolated. Cln-3.1 deletion mutants have a decreased lifespan, and cln-3.2 deletion mutants a decreased brood size. However, the neuronal or movement defects and aberrant lipopigment distribution or accumulation observed in JNCL were not found in the worms. To detect possible redundancy, single deletion mutants were crossed to obtain double and triple mutants, which were viable but showed no JNCL-specific defects. The cln-3 triple mutants show a more prominent decrease in lifespan and brood size, the latter most conspicuously at the end of the egg-laying period, suggesting premature ageing. To focus our functional analysis we examined the C. elegans cln-3 expression patterns, using promoter-GFP (green fluorescent protein) gene fusions. Fluorescence patterns suggest cln-3.1 expression in the intestine, cln-3.2 expression in the hypoderm, and cln-3.3 expression in intestinal muscle, male-specific posterior muscle and hypoderm. Further life stage- and tissue-specific analysis of the processes causing the phenotype of the cln-3 triple mutants may provide more information about the function of the cln-3 protein and contribute to a better understanding of the basic processes affected in Batten disease patients.

Genetic and morphological characterization of ftsB and nrdB mutants of Escherichia coli.

The ftsB gene of Escherichia coli is believed to be involved in cell division. In this report, we show that plasmids containing the nrdB gene could complement the ftsB mutation, suggesting that ftsB is an allele of nrdB. We compared changes in the cell shape of isogenic nrdA, nrdB, ftsB, and pbpB strains at permissive and restrictive temperatures. Although in rich medium all strains produced filaments at the restrictive temperature, in minimal medium only a 50 to 100% increase in mean cell mass occurred in the nrdA, nrdB, and ftsB strains. The typical pbpB cell division mutant also formed long filaments at low growth rates. Visualization of nucleoid structure by fluorescence microscopy demonstrated that nucleoid segregation was affected by nrdA, nrdB, and ftsB mutations at the restrictive temperature. Measurements of beta-galactosidase activity in lambda p(sfiA::lac) lysogenic nrdA, nrdB, and ftsB mutants in rich medium at the restrictive temperature showed that filamentation in the nrdA mutant was caused by sfiA (sulA) induction, while filamentation in nrdB and ftsB mutants was sfiA independent, suggesting an SOS-independent inhibition of cell division.

Cross-species homology of the CLN3 gene.

A murine cDNA clone was isolated by screening a mouse cDNA library with the human CLN3 cDNA. Sequence analysis indicates that the corresponding CLN3 proteins are highly homologous. We have compared these with recently identified CLN3 sequences from the dog, the nematode C. elegans, and baker's yeast S. cerevisiae. The CLN3 protein is remarkably conserved across eukaryotic species. Several protein modification sites which may be crucial for the function of the protein are conserved.

Rapid detection of the major deletion in the Batten disease gene CLN3 by allele specific PCR.

The recent isolation of the CLN3 gene involved in Batten disease (juvenile neuronal ceroid lipofuscinosis) creates possibilities for direct detection of mutations which can confirm or indicate the clinical diagnosis of Batten disease. We have designed a rapid and reliable allele specific PCR test for the detection of the major deletion, which can be used in carrier diagnosis, presymptomatic diagnosis, and prenatal diagnosis.

Replication of an incomplete alfalfa mosaic virus genome in plants transformed with viral replicase genes.

RNAs 1 and 2 of alfalfa mosaic virus (AIMV) encode proteins P1 and P2, respectively, both of which have a putative role in viral RNA replication. Tobacco plants were transformed with DNA copies of RNA1 (P1-plants), RNA2 (P2-plants) or a combination of these two cDNAs (P12-plants). All transgenic plants were susceptible to infection with the complete AIMV genome (RNAs 1, 2, and 3). Inoculation with incomplete mixtures of AIMV RNAs showed that the P1-plants were able to replicate RNAs 2 and 3, that the P2-plants were able to replicate RNAs 1 and 3, and that the P12-plants were able to replicate RNA3. Initiation of infection of nontransgenic plants, P1-plants, or P2-plants requires the presence of AIMV coat protein in the inoculum, but no coat protein was required to initiate infection of P12-plants with RNA3. Results obtained with P12-protoplasts supported the conclusion that coat protein plays an essential role in the replication cycle of AIMV RNAs 1 and 2.