Mutations in the RYR1 gene in Italian patients at risk for malignant hyperthermia: evidence for a cluster of novel mutations in the C-terminal region

Mutations in the ryanodine receptor type 1 (RYR1) gene are associated with Malignant Hyperthermia (MH) and Central Core Disease (CCD). We report here on the molecular analysis of the RYR1 gene in Italian families referred as potential cases of MH or in patients with CCD or multicore/minicore myopathy. Of a total of 20 individuals with mutations in the RYR1 gene, 14 were part of a group of 47 MH susceptible (MHS) patients, 4 of 34 individuals diagnosed as MH equivocal (MHE), and 2 were patients diagnosed with minicore myopathy and CCD, respectively. Mutations were found to segregate with the MHS or MHE phenotype within the families of the probands. A discordance between phenotype and genotype was observed in a family where a mutation detected in an MHS proband was also found in the father who had been diagnosed MH normal (MHN) at the IVCT. In addition to known mutations, seven novel mutations were found, five of which occurred in exons encoding the C-terminal region of RYR1. These results indicate that the C-terminal region of RYR1 represents an additional hot spot for mutations in patients with MH, similar to what has been reported for patients with CCD.     

Polymorphisms and deduced amino acid substitutions in the coding sequence of the ryanodine receptor (RYR1) gene in individuals with malignant hyperthermia.

Twenty-one polymorphic sequence variants of the RYR1 gene, including 13 restriction fragment length polymorphisms (RFLPs), were identified by sequence analysis of human ryanodine receptor (RYR1) cDNAs from three individuals predisposed to malignant hyperthermia (MH). All RFLPs were detectable in PCR-amplified products, and their segregation was consistent with our initial finding of linkage to MH in the nine families previously informative for one or more intragenic markers (MacLennan et al., 1990, Nature 343:559-561). Four amino acid substitutions were identified in the study: Arg for Gly248, Cys for Arg470, Leu for Pro1785, and Cys for Gly2059. Of 45 families tested, a single family presented the Arg for Gly248 substitution where it segregated with malignant hyperthermia, making it a candidate mutation for predisposition to MH in man. The other three polymorphic substitutions failed to segregate with malignant hyperthermia in those families in which they occurred, implying that they represent polymorphisms with little or no effect on the function of the RYR1 gene.     

Identification and analysis of mutations in the Wilson disease gene (ATP7B): population frequencies, genotype-phenotype correlation, and functional analyses.

Wilson disease (WD) is an autosomal recessive disorder characterized by toxic accumulation of copper in the liver and subsequently in the brain and other organs. On the basis of sequence homology to known genes, the WD gene (ATP7B) appears to be a copper-transporting P-type ATPase. A search for ATP7B mutations in WD patients from five population samples, including 109 North American patients, revealed 27 distinct mutations, 18 of which are novel. A composite of published findings shows missense mutations in all exons-except in exons 1-5, which encode the six copper-binding motifs, and in exon 21, which spans the carboxy-terminus and the poly(A) tail. Over one-half of all WD mutations occur only rarely in any population sample. A splice-site mutation in exon 12 accounts for 3% of the WD mutations in our sample and produces an in-frame, 39-bp insertion in mRNA of patients homozygous, but not heterozygous, for the mutation. The most common WD mutation (His1069Glu) was represented in approximately 38% of all the WD chromosomes from the North American, Russian, and Swedish samples. In several population cohorts, this mutation deviated from Hardy-Weinberg equilibrium, with an overrepresentation of homozygotes. We did not find a significant correlation between His1069Glu homozygosity and several clinical indices, including age of onset, clinical manifestation, ceruloplasmin activity, hepatic copper levels, and the presence of Kayser-Fleischer rings. Finally, lymphoblast cell lines from individuals homozygous for His1069Glu and 4 other mutations all demonstrated significantly decreased copper-stimulated ATPase activity.     

Single-amino-acid deletion in the RYR1 gene, associated with malignant hyperthermia susceptibility and unusual contraction phenotype

Malignant hyperthermia (MH) is an anesthetic-drug-induced, life-threatening hypermetabolic syndrome caused by abnormal calcium regulation in skeletal muscle. Often inherited as an autosomal dominant trait, MH has linkage to 30 different mutations in the RYR1 gene, which encodes a calcium-release-channel protein found in the sarcoplasmic reticulum membrane in skeletal muscle. All published RYR1 mutations exclusively represent single-nucleotide changes. The present report documents, in exon 44 of RYR1 in two unrelated, MH-susceptible families, a 3-bp deletion that results in deletion of a conserved glutamic acid at position 2347. This is the first deletion, in RYR1, found to be associated with MH susceptibility. MH susceptibility was confirmed among some family members by in vitro diagnostic pharmacological contracture testing of biopsied skeletal muscle. Although a single-amino-acid deletion appears to be a subtle change in the protein, the deletion of Glu2347 from RYR1 produces an unusually large electrically evoked contraction tension in MH-positive individuals, suggesting that this deletion produces an alteration in skeletal-muscle calcium regulation, even in the absence of pharmacological agents.     

The G1021A substitution in the RYR1 gene does not cosegregate with malignant hyperthermia susceptibility in a British pedigree.

A single base change in the RYR1 gene encoding the skeletal muscle ryanodine receptor (calcium-sensitive calcium-release channel of the sarcoplasmic reticulum), resulting in the substitution of G1021 by A, has been proposed to underlie malignant-hyperthermia (MH) susceptibility in as many as 10% of cases in the European population. As part of our mutation-screening program in MH-susceptible (MHS) individuals, we have investigated this substitution in individuals from 151 unrelated British MHS families and have detected G1021A heterozygotes in 7 families. This mutation was not found in 156 unrelated MH-negative (MHN) individuals. We also examined eight families with central core disease (CCD): the mutation did not occur in any family members of any disease status (affected or unaffected for CCD, MHS, or MHN). In one large family, the G1021A mutation was found but did not show complete cosegregation with MH susceptibility: it occurred in only 7/12 MHS individuals in the kinship, and susceptibility was inherited from parents who were G1021 homozygotes, as well as from parents who were heterozygotes. On the basis of these findings, it is clearly unreliable at present to offer presymptomatic DNA testing for MH status, even in families in which a mutation has been detected.     

Identification of novel mutations in the human EXT1 tumor suppressor gene

Hereditary multiple exostoses (EXT) is a genetically heterogeneous bone disorder caused by genes segregating on human chromosomes 8, 11, and 19 and designated EXT1, EXT2 and EXT3, respectively. Recently, the EXT1 gene has been isolated and partially characterized and appears to encode a tumor suppressor gene. We have identified six mutations in the human EXT1 gene from six unrelated multiple exostoses families segregating for the EXT gene on chromosome 8. One of the mutations we detected is the same 1-bp deletion in exon 6 that was previously reported in two independent EXT families. The other five mutations, in exons 1, 6, 9, and the splice junction at the 3′ end of exon 2, are novel. In each case, the mutation is likely to result in a truncated or nonfunctional EXT1 protein. These results corroborate and extend the previous report of mutations in this gene in two EXT families, and provide additional support for the EXT1 gene as the cause of hereditary multiple exostoses in families showing linkage to chromosome 8. Received: 2 August 1996 / Revised: 18 November 1996     

肌萎缩侧索硬化患者SOD1基因突变检测及突变与临床表型的关系

应用PCR技术结合DNA直接测序方法对8例临床确诊为家族性肌萎缩侧索硬化(Familiar amyotrophic lateral sclerosis,FALS)家系的先证者进行铜锌超氧化物歧化酶基因(SOD1)的突变筛查,在3例先证者中检出2种SOD1基因突变,其中,2例携带了位于4号外显子的错义突变Cys111Tyr(c.332G>A),另1例携带了位于5号外显子的错义突变Gly147Asp(c.440G>A),这2种突变在中国ALS患者中属首次报道。该结果扩大了中国FALS患者的SOD1基因突变谱,对研究中国FALS患者SOD1基因突变特点和分布规律有一定帮助。分析携带这2个突变患者的临床特点,提示Cys111Tyr突变导致的临床表型相对温和,而Gly147Asp突变可导致病情进展较快。该结果有待在更多的病例中进行证实。     

A genotype-phenotype correlation between null-allele mutations in the ferrochelatase gene and liver complication in patients with erythropoietic protoporphyria.

Erythropoietic protoporphyria (EPP), an inborn error of heme metabolism, causes in the majority of the patients only a symptom of photosensitivity. However, around 2% of the EPP sufferers develop liver complication in the form of liver cirrhosis and progressive liver failure. Mutations in the human ferrochelatase (FECH) gene causing EPP are highly heterogeneous and mostly family-specific. Actually, 62 FECH mutations have been published, 48 of them are "null allele" mutations inducing the formation of a truncated protein. The remaining 14 are missense mutations. In contrast to the null allele mutations, the latter lead to substitution of a single amino acid residue in the protein molecule and generate an enzyme that, although functionally impaired, is in its full length. In order to study the association between "null allele" mutation and liver complication, we combined our data with those in the literature. A total of 112 EPP patients were counted among 93 EPP families with a known FECH mutation. All 18 EPP patients who had severe liver complication carried a "null allele" mutation. In contrast, none of the 20 patients who carried a missense mutation had developed liver complication till the time of study (Fisher's exact test, p<0.05). High protoporphyrin blood concentration are considered to be a sign of an increased risk of liver disease. No correlation of protoporphyrin blood level with the type of mutation, was found, if patients with overt liver disease were excluded from the sample. Furthermore, no significant association of the liver complication with the location of the mutation within the FECH gene was found (Fisher exact test p = 0.46). These available data indicate a significant genotype-phenotype correlation between "null allele" mutation and protoporphyrin related liver disease in EPP. Although the risk for a EPP patient with a missense mutation to develop liver disease cannot be totally eliminated based on these data, it is comparably low.     

Identification of three novel mutations in the PIG-A gene in paroxysmal nocturnal haemoglobinuria (PNH) patients

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired haemolytic disorder caused by the absence of glycosyl phosphatidylinositol (GPI)-anchored surface proteins resulting from a defect in one step of GPI-anchor biosynthesis. Recent analysis has shown that mutations at the PIG-A (phosphatidylinositoglycan-class A) gene are responsible for GPI-anchor deficiency in all PNH patients. In the current study, we describe three new mutations of the PIG-A gene in Italian patients with PNH. The analysis has been performed by RNA/single-strand conformation polymorphism using genomic DNA purified from nucleated peripheral blood cells. An abnormal pattern of migration of polymerase chain reaction amplified fragments containing exons 2 and 5 was observed. Sequencing analysis led to the identification of three mutations: a transversion C-to-A creating a stop codon (Y98X), an A insertion at position 460 (460insA), and a C deletion (1114delC). All the mutations cause a premature termination of the translation of the PIG-A protein.     

Identification of novel mutations in L1CAM gene by a DHPLC-based assay

X-linked hydrocephalus, MASA syndrome, X-linked complicated Spastic Paraplegia Type I, and X-linked partial agenesis of the corpus callosum are rare diseases mainly affecting male population and broadly referred as L1 syndrome, caused by mutations in the L1CAM gene. In the present study 36 boys and a male fetus whose clinical features were consistent with L1 syndrome were analyzed by dHPLC assay and direct sequencing of L1CAM gene. Sequence analysis of the 14 different aberrant dHPLC elution profiles demonstrated that six of them were associated with already reported polymorphisms, four with previously described causative variants while the remaining four represented novel L1CAM mutations. The dHPLC method proposed identified eight (21 %) causative L1CAM mutations in our patients while direct sequencing failed to detect any variation in patients negative to dHPLC analysis. We conclude that the dHPLC assay represents a fast and efficient method for the screening of L1CAM mutations and that L1 syndrome should be considered in the differential diagnosis of intellectual disability in children, especially when other signs such as hydrocephalus or adducted thumbs are present.     

Myotonia congenita: novel mutations in CLCN1 gene

Myotonia congenita belongs to the group of non-dystrophic myotonia caused by mutations of CLCN1gene, which encodes human skeletal muscle chloride channel 1. It can be inherited either in autosomal dominant (Thomsen disease) or recessive (Becker disease) forms. Here we have sequenced all 23 exons and exon-intron boundaries of the CLCN1 gene, in a panel of 5 unrelated Chinese patients with myotonia congenita (2 with dominant and 3 with recessive form). In addition, detailed clinical analysis was performed in these patients to summarize their clinical characteristics in relation to their genotypes. Mutational analyses revealed 7 different point mutations. Of these, we have found 3 novel mutations including 2 missense (R47W, V229M), one splicing (IVS19+2T>C), and 4 known mutations (Y261C,G523D, M560T, G859D). Our data expand the spectrum of CLCN1 mutations and provide insights for genotype–phenotype correlations of myotonia congenita in the Chinese population.     

Myotonia congenita: novel mutations in CLCN1 gene

Myotonia congenita belongs to the group of non-dystrophic myotonia caused by mutations of CLCN1gene, which encodes human skeletal muscle chloride channel 1. It can be inherited either in autosomal dominant (Thomsen disease) or recessive (Becker disease) forms. Here we have sequenced all 23 exons and exon-intron boundaries of the CLCN1 gene, in a panel of 5 unrelated Chinese patients with myotonia congenita (2 with dominant and 3 with recessive form). In addition, detailed clinical analysis was performed in these patients to summarize their clinical characteristics in relation to their genotypes. Mutational analyses revealed 7 different point mutations. Of these, we have found 3 novel mutations including 2 missense (R47W, V229M), one splicing (IVS19+2T>C), and 4 known mutations (Y261C,G523D, M560T, G859D). Our data expand the spectrum of CLCN1 mutations and provide insights for genotype–phenotype correlations of myotonia congenita in the Chinese population.     

Identification of novel mutations of the DAX-1 gene in patients with X-linked adrenal hypoplasia congenita

OBJECTIVE: X-linked adrenal hypoplasia congenita (AHC) is a condition clinically featuring adrenal insufficiency and hypogonadotropic hypogonadism caused by mutations of DAX-1. This study was undertaken to characterize the molecular defects of DAX-1 in 3 unrelated Korean patients with AHC. PATIENTS AND METHODS: Patient 1 is a 6-year-old boy who presented with a salt-losing adrenal crisis in the neonatal period. Patient 2 is a 3-year-old boy who manifested aspiration pneumonia and adrenal insufficiency at the age of 1 month. Patient 3 is a 7-year-old boy who developed an adrenal crisis at the age of 3 days. In each of these patients, DAX-1 was analyzed by direct DNA sequencing after polymerase chain reaction amplification of the entire coding region. RESULTS: Direct sequencing of DAX-1 revealed two novel mutations, 1156_1157delCT in patient 1 and another novel nonsense mutation W105X in patient 2. Patient 3 had complete deletion of DAX-1. In patient 3, serum transaminases and creatine kinase levels were elevated while the glycerol kinase activity of leukocytes was decreased. Markedly elevated glycerol excretion was detected by urine organic acid analysis. Patient 3 was diagnosed as Xp21 contiguous gene syndrome associated with deletions of the entire IL1RAPL, GK genes and the C-terminal region of DMD gene. CONCLUSIONS: Two novel mutations of DAX-1 were detected in 2 unrelated patients with AHC, and complete deletion of DAX-1 in a patient with Xp21 contiguous gene syndrome who also presented with glycerol kinase deficiency, Duchenne muscular dystrophy, and AHC.     

Small supernumerary marker chromosomes (SMCs): genotype-phenotype correlation and classification

Small supernumerary marker chromosomes (SMCs) are present in about 0.05% of the human population. In approximately 30% of SMC carriers (excluding the ~60% SMC derived from one of the acrocentric chromosomes), an abnormal phenotype is observed. The clinical outcome of an SMC is difficult to predict as they can have different phenotypic consequences because of (1) differences in euchromatic DNA-content, (2) different degrees of mosaicism, and/or (3) uniparental disomy (UPD) of the chromosomes homologous to the SMC. Here, we present 35 SMCs, which are derived from all human chromosomes, apart from chromosome 6, as demonstrated by the appropriate molecular cytogenetic approaches, such as centromere-specific multicolor fluoresence in situ hybridization (cenM-FISH), multicolor banding (MCB), and subcentromere-specific multicolor FISH (subcenM-FISH). In nine cases without an aberrant phenotype, neither partial proximal trisomies nor UPD could be detected. Abnormal clinical findings, such as psychomotoric retardation and/or craniofacial dysmorphisms, were associated with seven of the cases in which subcentromeric single-copy probes were proven to be present in three copies. Conversely, in eight cases with a normal phenotype, proximal euchromatic material was detected as partial trisomy. UPD was studied in 12 cases and subsequently detected in two of the cases with SMC (partial UPD 4p and maternal UPD 22 in a der(22)-syndrome patient), indicating that SMC carriers have an enhanced risk for UPD. At present, small proximal trisomies of 1p, 1q, 2p, 6p, 6q, 7q, 9p, and 12q seem to lead to clinical manifestations, whereas partial proximal trisomies of 2q, 3p, 3q, 5q, 7p, 8p, 17p, and 18p may not be associated with significant clinical symptoms. With respect to clinical outcome, a classification of SMCs is proposed that considers molecular genetic and molecular cytogenetic characteristics as demonstrated by presently available methods.Electronic database information: accession numbers and URLs for the data in this article are as follows:ENSEMBL-database, National Center for Biotechnology Information (NCBI), Genome Database (GDB), OMIM (Online Mendelian Inheritance in Man) Database,     

The roles of two novel FBN1 gene mutations in the genotype-phenotype correlations of Marfan syndrome and ectopia lentis patients with marfanoid habitus

Mutations in the fibrillin-1 (FBN1) gene have been identified in patients with Marfan syndrome (MFS) and Marfan-like connective tissue disorders. In this study, two Chinese families were recruited. The patients in family 1 were well characterized with MFS, while those in family 2 displayed Marfan-like disorders such as ectopia lentis (EL) and marfanoid habitus, but did not develop cardiovascular diseases. We aimed to analyze the pathogenic mutations and their relationships with phenotypes in these two Chinese families. All participants underwent complete physical, ophthalmic, and cardiovascular examinations. The 65 exons and flanking intronic sequences of FBN1 were amplified by polymerase chain reaction, and screened for mutations by denaturing high-performance liquid chromatography and sequencing. One hundred and fifteen unrelated controls were analyzed using the same methods to confirm the mutations. In family 1, we identified the mutation p.C499S in the calcium-binding epidermal growth factor (cbEGF)-like domain 3 of FBN1. In family 2, the mutation p.C908Y was identified in an interdomain region of the hybrid motif 2 linked to the cbEGF-like domain 10. It can be concluded that FBN1 mutations involving cysteine substitutions are usually associated with MFS and EL with some MFS features. Moreover, pathology seemed more serious when the mutations disrupted the three disulfide bridges in the cbEGF-like domains, which was more likely to cause typical MFS than if the mutations occurred in the hybrid motifs. Our data preliminarily establish a genotype-phenotype correlation in the diagnostic process of MFS and predominant EL with Marfan-like features.     

Identification of mutations in exons 1 through 8 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

Five different mutations have been identified in the gene causing cystic fibrosis (CF) through sequencing regions encompassing exons 1-8, including the 5' untranslated leader. Two of these apparent mutations are missense mutations, one in exon 3 (Gly to Glu at position 85; G85E) and another in exon 5 (Gly to Arg at 178; G178R), both causing significant changes in the corresponding amino acids in the encoded protein--cystic fibrosis transmembrane conductance regulator (CFTR). Two others affect the highly conserved RNA splice junction flanking the 3' end of exons 4 and 5 (621 + 1G----T, 711 + 1G----T), resulting in a probable splicing defect. The last mutation is a single-basepair deletion in exon 4, causing a frameshift. These five mutations account for the 9 of 31 non-delta F508 CF chromosomes in our Canadian CF family collection and they are not found in any of the normal chromosomes. Three of the mutations, 621 + 1G----T, 711 + 1G----T, and G85E, are found in the French-Canadian population, with 621 + 1G----T being the most abundant (5/7). There are two other sequence variations in the CFTR gene; one of them (129G----C) is located 4 nucleotides upstream of the proposed translation initiation codon and, although present only on CF chromosomes, it is not clear whether it is a disease-causing mutation; the other (R75Q) is most likely a sequence variation within the coding region.