Eukaryotic peptide chain release factor 1 participates in translation termination of specific cysteine-poor prolamines in rice endosperm.

Prolamines are alcohol-soluble proteins classified as either cysteine-poor (CysP) or cysteine-rich (CysR) based on whether they can be alcohol-extracted without or with reducing agents, respectively. In rice esp1 mutants, various CysP prolamines exhibit both reduced and normal amounts of isoelectric focusing bands, indicating that the mutation affects only certain prolamine classes. To examine the genetic regulation of CysP prolamine synthesis and accumulation, we constructed a high-resolution genetic linkage map of ESP1. The ESP1 gene was mapped to within a 20 kb region on rice chromosome 7. Sequencing analysis of annotated genes in this region revealed a single-nucleotide polymorphism within eukaryotic peptide chain release factor (eRF1), which participates in stop-codon recognition and nascent-polypeptide release from ribosomes during translation. A subsequent complementation test revealed that ESP1 encodes eRF1. We also identified UAA as the stop codon of CysP prolamines with reduced concentration in esp1 mutants. Recognition assays and microarray analysis confirmed that ESP1/eRF1 recognizes UAA/UAG, but not UGA. Our results provide convincing evidence that ESP1/eRF1 participates in the translation termination of CysP prolamines during seed development.

Homozygosity mapping with SNP arrays confirms 3p21 as a recessive locus for gray platelet syndrome and narrows the interval significantly.

Gray platelet syndrome (GPS) is an inherited bleeding disorder characterized by thrombocytopenia and the absence of α-granules in platelets. Patients with GPS present with mild to moderate bleeding and many develop myelofibrosis. The genetic cause of GPS is unknown. We present 2 Native American families with a total of 5 affected persons and a single affected patient of Pakistani origin in which GPS appears to be inherited in an autosomal recessive manner. Homozygosity mapping using the Affymetrix 6.0 chips demonstrates that all 6 GPS-affected persons studied are homozygous for a 1.7-Mb region in 3p21. Linkage analysis confirmed the region with a logarithm of the odds score of 2.7. Data from our families enabled us to significantly decrease the size of the critical region for GPS from the previously reported 9.4-Mb region at 3p21.

Mapping of autosomal dominant cerebellar ataxia without the pathogenic PPP2R2B mutation to the locus for spinocerebellar ataxia 12.

OBJECTIVES: To map the disease locus and to identify a gene mutation in a Japanese family with autosomal dominant cerebellar ataxia. DESIGN: A genome-wide linkage analysis was performed using the Affymetrix genome-wide human single-nucleotide polymorphism array containing 909 622 single-nucleotide polymorphisms. Direct nucleotide sequencing of a candidate gene was performed. SETTING: Hokkaido University Graduate School of Medicine and Tokyo University Graduate School of Medicine. Patients  Four affected and 6 healthy individuals in a family with autosomal dominant cerebellar ataxia. RESULTS: One locus on chromosome 5q had a multipoint logarithm of odds score of 2.408, the theoretical maximum. This locus was flanked by markers rs681591 and rs32582 and includes PPP2R2B (protein phosphatase 2, regulatory subunit B, beta isoform), the causative gene of autosomal dominant spinocerebellar ataxia 12 (SCA12). However, unlike SCA12, no CAG repeat expansions in the promoter region and no nucleotide substitution or insertion-deletion mutations in the exons of the PPP2R2B gene were found. CONCLUSION: Autosomal dominant cerebellar ataxia mapping to 5q31-q33.1 has no CAG repeat expansion or other mutations of the PPP2R2B gene.

Amyotrophic lateral sclerosis linked to a novel SOD1 mutation with muscle mitochondrial dysfunction.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative motor neuron disorder. Mutations in Cu,Zn superoxide dismutase (SOD1) cause approximately 20% of familial ALS. One of the possible mechanisms whereby they induce disease is mitochondrial dysfunction in motor neurons. Here we describe a patient with ALS and muscle mitochondrial oxidative defect associated with a novel SOD1 mutation. Direct sequencing of SOD1 gene revealed a heterozygous mutation in codon 22 substituting a highly conserved amino acid, from glutamine to arginine (Q22R). Muscle biopsy showed a neurogenic pattern associated with cytochrome c oxidase (COX) deficiency in several muscle fibers. Western blot analysis demonstrated a reduction in SOD1 content in the cytoplasmic and mitochondrial fractions. These results suggest that a minute quantity of mutant SOD1 protein contributes to a mitochondrial toxicity also in muscle tissue.

Microsatellite analysis of the GLC1B locus on chromosome 2 points to NCK2 as a new candidate gene for normal tension glaucoma.

AIMS: The aim of this study was to investigate the association between normal tension glaucoma and the candidate disease locus glaucoma 1, open angle, B (GLC1B) on chromosome 2. There are many reports describing the results of association or linkage studies for primary open angle glaucoma (POAG), with GLC1B as one of the loci associated with normal or moderately elevated intraocular pressure. However, there are few reports about the association of genes or defined genomic regions with normal tension glaucoma, which is the leading type of glaucoma in Japan. The GLC1B locus is hypothesized to be a causative region for normal tension glaucoma. METHODS: Genomic DNA was extracted from whole blood of normal tension glaucoma (n = 143) and healthy controls (n = 103) of Japanese origin. RESULTS: Fifteen microsatellite markers within and/or near to the GLC1B locus were genotyped, and their association with normal tension glaucoma was analysed. Two markers D2S2264 and D2S176 had significant positive associations. CONCLUSION: The D2S176 marker had the strongest significant association and it is located 24 kb from the nearest gene NCK2, which now becomes an important new candidate gene for future studies of its association with normal tension glaucoma.

Familial epilepsy and developmental dysphasia: description of an Italian pedigree with autosomal dominant inheritance and screening of candidate loci.

PURPOSE: To describe a familial epileptic condition combining a peculiar electro-clinical pattern with developmental language dysfunction in a large Italian kindred. METHODS: We studied the clinical and neurophysiological features of a 4-generation family with 10 affected members (3 deceased). We also analysed in 7 affected and 7 healthy members microsatellite markers for 51 candidate loci for epilepsy, including 42 loci containing ion channel genes expressed in the brain, as well as the SPCH1 and SRPX2 loci. RESULTS: Five of the seven living affected members (aged 20-58 years) had the full phenotype (seizures, EEG epileptiform abnormalities and dysphasia). The language dysfunction was the first symptom, becoming evident since the period of language development and mainly consisting of phonemic and syntactic paraphasias, difficulty of expression and reduced verbal fluency. The seizures had their onset between 2 and 23 years and were reported as epileptic falls (4) associated or not with myoclonic features, absences (3), tonic-clonic (1) and complex partial seizures (1). The seizures were easily controlled by antiepileptic treatment in all patients except one. In the five patients with a good response of seizures to treatment, the EEG tracings showed the coexistence of focal and generalized epileptiform abnormalities; in the refractory patient the interictal EEG demonstrated bilateral asynchronous fronto-temporal paroxysms with left predominance and ictal SEEG recording suggested a multifocal origin of the discharges. MRI of the brain was normal in all patients. Linkage analysis provided negative LOD scores for all the investigated loci. CONCLUSION: We have described a novel familial pattern of epilepsy and developmental dysphasia which is not genetically linked to epilepsy or speech disorder loci, as documented by a candidate-gene linkage approach.

Reduced regional cerebral blood flow in SPG4-linked hereditary spastic paraplegia.

Hereditary spastic paraplegia (HSP) linked to the spastic gait gene 4 (SPG4) is controversial, as the "pure" form traditionally has been considered confined to the long axons of the spinal cord. However, recent immunolabeling experiments have demonstrated extensive Spastin expression in the cortex and striatum. This could indicate a more widespread neuropathology from mutations in the SPG4 gene than previously assumed. The aim of this study was therefore to ascertain the extent of cerebral involvement in SPG4 linked HSP by neuropsychological examination and measurement of the regional cerebral blood flow (rCBF) as an indirect marker of regional neuronal activity. Eighteen SPG4 patients and 18 matched control subjects were studied. Resting state rCBF was measured using Positron Emission Tomography (PET) and the (15)O-labelled water bolus technique and relative group differences were explored using Statistical Parametric Mapping (SPM 99). Neuropsychological assessment was performed using established and nationally validated tests (RH Basic Battery). Compared to healthy controls, the patient group had significantly decreased rCBF in the left fronto-temporal cortex (P<0.05), and more extensive changes were observed in a separate analysis of the most disabled individuals. The neuropsychological assessment revealed only significantly impaired recognition memory for faces. In summary, the findings support cerebral pathology in SPG4-linked HSP, although the decreased rCBF in fronto-temporal cortex was not associated with severe cognitive impairment.

The molecular-genetic basis of functional hyperandrogenism and the polycystic ovary syndrome.

The genetic mechanisms underlying functional hyperandrogenism and the polycystic ovary syndrome (PCOS) remain largely unknown. Given the large number of genetic variants found in association with these disorders, the emerging picture is that of a complex multigenic trait in which environmental influences play an important role in the expression of the hyperandrogenic phenotype. Among others, genomic variants in genes related to the regulation of androgen biosynthesis and function, insulin resistance, and the metabolic syndrome, and proinflammatory genotypes may be involved in the genetic predisposition to functional hyperandrogenism and PCOS. The elucidation of the molecular genetic basis of these disorders has been burdened by the heterogeneity in the diagnostic criteria used to define PCOS, the limited sample size of the studies conducted to date, and the lack of precision in the identification of ethnic and environmental factors that trigger the development of hyperandrogenic disorders. Progress in this area requires adequately sized multicenter collaborative studies after standardization of the diagnostic criteria used to classify hyperandrogenic patients, in whom modifying environmental factors such as ethnicity, diet, and lifestyle are identified with precision. In addition to classic molecular genetic techniques such as linkage analysis in the form of a whole-genome scan and large case-control studies, promising genomic and proteomic approaches will be paramount to our understanding of the pathogenesis of functional hyperandrogenism and PCOS, allowing a more precise prevention, diagnosis, and treatment of these prevalent disorders.

The angiotensin II receptor (Agtr1a): functional regulatory polymorphisms in a locus genetically linked to blood pressure variation in the mouse.

Hypertension is a complex trait with multiple genetic determinants. A previous genome-wide linkage study of systolic blood pressure in a mouse genetic backcross implicated a region of chromosome 13 (LOD = 3.3 at 16.0 cM) as a determinant of blood pressure differences between a hereditary low blood pressure strain of Mus musculus (BPL/1) and Mus spretus (SPRET); at this locus, the unexpected effect of the BPL/1 allele was to increase blood pressure. A plausible candidate locus encoding angiotensin II receptor isoform 1a (Agtr1a) is also located at 16.0 cM on chromosome 13. We therefore investigated structural and functional differences at Agtr1a between BPL/1 and SPRET, as well as the BPH/2 strain. Resequencing Agtr1a in the three strains established the exon/intron and proximal promoter structure of the mouse gene. Coding exon 3 spanned 1,960 bp (with 26 SNPs), including the 1,077-bp/359-amino acid ORF (with 5 cSNPs, all of which were synonymous). Promoter sequences revealed a consensus TATA box, conserved G/C-rich regions, and a striking, lengthy simple sequence repeat region, composed of di-, tri-, tetra-, and penta-nucleotide repeats, whose overall length varied markedly among the strains. Twenty-five other SNPs and three single nucleotide deletions differentiated the strains' promoters, six of which were in likely functional promoter motifs. Agtr1a mRNA abundance in the adrenal gland in vivo was greater (P < 0.05) in BPL/1 than SPRET, consistent with the predicted effect of the BPL/1 allele to confer higher blood pressure. When Agtr1a promoters were subcloned into luciferase reporter plasmids and transfected into PC12 chromaffin cells, basal promoter expression was higher (P < 0.001) in BPL/1 than in SPRET, consistent with the endogenous mRNA results. In summary, Agtr1a on chromosome 13 is highly polymorphic between mouse strains, although the amino acid sequence specified by the ORF is invariant, even across mouse species. We conclude that polymorphisms in the Agtr1a promoter account for differences in gene expression in vivo between BPL/1 and SPRET, in a way consistent with the effects of alleles at this locus on chromosome 13 to affect blood pressure in the mouse genome-wide linkage study.

Linkage for platelet monoamine oxidase (MAO) activity: results from a replication sample.

BACKGROUND: Monoamine oxidase B (MAO-B) degrades catecholamines in presynaptic nerve endings and is also active in platelets. There is evidence to suggest that platelet MAO-B activity level is controlled by a major genetic locus distinct from the structural gene on the X chromosome. To expand on a prior report, new linkage analyses for platelet MAO-B activity have been performed on the previously analyzed sample (designated the initial sample), on a new sample of families (the replication sample), and on the combined sample. These families were recruited as part of the Collaborative Study on the Genetics of Alcoholism (COGA). METHODS: The initial sample consists of 105 extended families providing 1002 nonindependent (412 independent) sib pairs that have been phenotyped for MAO activity and genotyped. The replication sample of 157 extended families contains 608 nonindependent (309 independent) phenotyped and genotyped sib pairs. Analyses were conducted using Haseman-Elston based regression on sib pairs and variance component analysis on extended pedigrees, and the importance of cigarette smoking and gender as covariates of platelet MAO-B activity was taken into account. RESULTS: Regions on chromosomes 2, 9, and 12 indicated consistent evidence for linkage across the two distinct datasets by at least one analysis method. Under Haseman-Elston regression of independent sib pairs, only the chromosome 2 region gave lod scores above 1 in both the initial and replication samples. Using all possible pairs, unweighted, for the regression, chromosome 12 gave lod scores above 1 in both samples. For variance component analysis, only the chromosome 9 region gave lod scores above 1 in both samples. CONCLUSIONS: The consistency across datasets of these findings is encouraging. In particular, variance component analysis of extended pedigrees supports a potential linkage of MAO-B activity to chromosome 9, with a lod over 3 at 115 cM near D9S261 in the combined sample. Sib-pair regression supports this finding with modest lod scores in the region. Suggestive linkage to chromosomes 2 and 12 from sib-pair analysis is only weakly supported by variance component analysis.

Familial calcium crystal diseases: what have we learned?

The spectrum of heterotopic calcification or ossification is expanding because of the reports of several kindreds with calcium pyrophosphate deposition disease, apatite deposition disease, and others with less common syndromes associated with extracellular matrix calcification, such as fibrodysplasia ossificans progressiva and related syndromes. Genomic DNA studies in both humans and mice provide a shortcut to understanding the genetic basis of promotion and prevention of ECM calcification. Mutation in the COL2A1 gene has been identified in one family with spondyloepiphyseal dysplasia and calcium pyrophosphate and apatite crystalline deposits. In another kindred with precocious osteoarthritis without spondyloepiphyseal dysplasia, the phenotype was linked to markers of chromosome 8. In four other kindreds, the phenotypes were linked to an area of chromosome 5p. Two genes located in this region, which are expressed in articular cartilage, are being investigated as possible calcium pyrophosphate deposition disease genes. The results of linkage studies in three kindreds with articular/periarticular ADD with the COL2A1 gene were noninformative. Two different mouse mutations, the ank/ank and the ttw/ttw mice, are associated with intra-articular and ligament apatite deposits caused by a decrease in extracellular pyrophosphate concentrations, mimicking human arthritis caused by apatite deposition disease. Mutations in the matrix GLA protein, both in mice and in humans, are also associated with vascular and articular calcification. These mouse mutations provide cutting-edge information in the investigation of the mechanisms of apatite deposition in humans.

A novel frameshift mutation in the McLeod syndrome gene in a Japanese family.

We report a novel mutation in the XK gene (XK) in a Japanese patient with McLeod syndrome. A 50-year-old man showed progressive muscular atrophy, choreic movement, elevated level of serum creatinine kinase, and acanthocytosis. The expression level of all the Kell antigens in erythrocyte was decreased and molecular analysis revealed a single-base (T) deletion at the nucleotide position 1095 in XK. This deletion caused a frameshift in translation, leading to a premature stop codon at the amino acid position 408. We conclude this single-base deletion causes defective Kx protein, which is responsible for the McLeod phenotype in this patient.

Molecular analysis of major histocompatibility complex allelic associations with systemic lupus erythematosus in Taiwan.

OBJECTIVE: To investigate the association of HLA class II alleles/haplotypes, type I C2 deficiency gene, and tumor necrosis factor a gene promoter allele (TNF2) with systemic lupus erythematosus (SLE) in the Chinese population in Taiwan. METHODS: The HLA-DRB1 and DQB1 alleles were studied in 105 SLE patients and 115 controls by the polymerase chain reaction (PCR)/sequence-specific oligonucleotide probe method, the subtyping of DRB1*15/16 and DRB5 by PCR with sequence-specific primers, type I C2 deficiency gene by PCR, and TNF2 by PCR-Nco I restriction fragment length polymorphism. RESULTS: The frequencies of the HLA class II alleles DRB1*02, DRB1*1502, DRB5*0102, DQB1*0501, and DQB1*0602 and DR2-associated haplotypes DRB1* 1501,DRB5*0101,DQB1*0602 and DRB1*1502,DRB5* 0102,DQB1*0501 were higher among SLE patients than among controls; however, only DQB1*0501 was statistically significantly associated with SLE. No specific allele/haplotype was significantly associated with lupus nephritis. No subject had type I C2 deficiency. SLE patients had a marginally higher percentage of TNF2, which was in linkage disequilibrium with DR3. Since DR3 was not associated with SLE in this Taiwanese Chinese population, TNF2 might play a role in the immunopathogenesis of SLE. CONCLUSION: Although no HLA-DRB1 allele was found to be significantly associated with SLE, the associations with DQB1*0501 and TNF2 suggest that DQB1 and tumor necrosis factor a may be important genetic factors in SLE susceptibility in the Chinese population in Taiwan.

Diagnosis of susceptibility to malignant hyperthermia with flanking DNA markers.

OBJECTIVE: To define the region on human chromosome 19 carrying the gene for malignant hyperthermia susceptibility and to evaluate the use of flanking DNA markers in diagnosing susceptibility. DESIGN: Prospective molecular genetic linkage studies in a large malignant hyperthermia pedigree. SETTING: Irish malignant hyperthermia testing centre. SUBJECTS: A large Irish malignant hyperthermia pedigree. MAIN OUTCOME MEASURES: Routine diagnosis of susceptibility to malignant hyperthermia with in vitro contracture test on muscle biopsy specimens and genetic linkage between susceptibility and polymorphic DNA markers in a malignant hyperthermia family. RESULTS: Genetic typing of polymorphic DNA markers in a large Irish malignant hyperthermia pedigree generated a lod score of greater than 3 for the marker D19S9 and showed that the gene for susceptibility is flanked by the markers D19S9 and D19S16. These tightly linked flanking markers allowed non-invasive presymptomatic diagnosis of susceptibility in five untested subjects in the large pedigree with an accuracy of greater than 99.7%. CONCLUSIONS: DNA markers flanking the gene for susceptibility to malignant hyperthermia can be used with high accuracy to diagnose susceptibility in subjects in large known malignant hyperthermia pedigrees and may replace the previous in vitro contracture test for diagnosing this inherited disorder in large families with malignant hyperthermia.

Genetic mechanisms of neoplasia in MEN 2.

Several possible mechanisms for the initiation and progression of tumors in multiple endocrine neoplasia type 2 (MEN 2) merit consideration. Localization of MEN2A to the pericentromeric area of chromosome 10 indicates the site of the initial mutagenic event but does not explain the tissue specificity observed. The consistency of tissue involvement within families, despite the variability between families, suggests that the tumors result from separate but contiguous tissue-specific genes arranged in a particular linear order. Linkage studies in MEN 2A and 2B families are compatible with this contiguous gene theory. Data suggest that Knudson's two-mutational-event theory is applicable in MEN 2, with cellular hyperplasia resulting from the initial heritable mutation. The second event could be a homozygous allelic mutation, but the lack of consistent loss of heterozygosity of chromosome 10 markers in tumors suggests other mechanisms. Observations in MEN 2 may be explained by the heritable chromosome 10 mutation causing hyperplasia, with the hyperplastic cells being converted to cancer cells by second mutations at any of many possible sites. Tumor progression probably involves subsequent events at other loci. These hypotheses may have important clinical implications.