Point mutation tRNA(Ser(UCN)) in a child with hearing loss and myoclonus epilepsy

We report on a family with a 12-year-old boy who suffered from a maternally inherited syndrome characterized by a combination of sensorineural hearing loss, myoclonus epilepsy, ataxia, severe psychomotor retardation, short stature, and diabetes mellitus. First, he showed a muscular hypotonia with hearing loss; later, he developed a myoclonus epilepsy, growth failure, and severe psychomotor retardation. At the age of 10 years, he developed diabetes mellitus. After initiation of combined ubiquinone and vitamin C treatment, we observed a progression in psychomotor development. Lactate and pyruvate levels in blood and cerebrospinal fluid were normal. No ragged red fibers or ultrastructural abnormalities were seen in a skeletal muscle biopsy. Biochemical assays of respiratory chain complex activities revealed decreased activity of complexes I and IV. By sequence analysis of mitochondrial DNA encoding transfer ribonucleic acids (RNAs), a homoplasmic T to C substitution at nucleotide position 7512 was found affecting a highly conserved base pair in the tRNA(ser(UCN)) acceptor stem. Asymptomatic family members of the maternal line were heteroplasmic for the mutation in blood samples. Analysis of mitochondrial DNA in patients with hearing loss and myoclonus epilepsy is recommended, even in the absence of laboratory findings. Therapeutically, ubiquinone and antioxidants can be beneficial.

MET Y1253D-activating point mutation and development of distant metastasis in advanced head and neck cancers

We investigated if the MET-activating point mutation Y1253D influences clinical outcomes in patients with advanced squamous cell carcinoma of the head and neck (HNSCC). The study population consisted of 152 HNSCC patients treated by hyperfractionated radiotherapy alone or concomitant with chemotherapy between September 1994 and July 2000. Tumors were screened for the presence of the MET-activating point mutation Y1253D. Seventy-eight patients (51%) received radiotherapy alone, 74 patients (49%) underwent radiotherapy concomitant with chemotherapy. Median patient age was 54 years and median follow-up was 5.5 years. Distant metastasis-free survival, local relapse-free survival and overall survival were compared with MET Y1253D status. During follow-up, 29 (19%) patients developed distant metastasis. MET Y1253D was detected in tumors of 21 out of 152 patients (14%). Distant metastasis-free survival (P = 0.008) was associated with MET Y1253D. In a multivariate Cox regression model, adjusted for T-category, only presence of MET Y1253D was associated with decreased distant metastasis-free survival: hazard ratio = 2.5 (95% confidence interval: 1.1, 5.8). The observed association between MET Y1253D-activating point mutation and decreased distant metastasis-free survival in advanced HNSCC suggests that MET may be a potential target for specific treatment interventions.

A point mutation in cpsE renders Streptococcus pneumoniae nonencapsulated and enhances its growth, adherence and competence

BackgroundThe polysaccharide capsule is a major virulence factor of the important human pathogen Streptococcus pneumoniae. However, S. pneumoniae strains lacking capsule do occur.ResultsHere, we report a nasopharyngeal isolate of Streptococcus pneumoniae composed of a mixture of two phenotypes; one encapsulated (serotype 18C) and the other nonencapsulated, determined by serotyping, electron microscopy and fluorescence isothiocyanate dextran exclusion assay.By whole genome sequencing, we demonstrated that the phenotypes differ by a single nucleotide base pair in capsular gene cpsE (C to G change at gene position 1135) predicted to result in amino acid change from arginine to glycine at position 379, located in the cytoplasmic, enzymatically active, region of this transmembrane protein. This SNP is responsible for loss of capsule production as the phenotype is transferred with the capsule operon. The nonencapsulated variant is superior in growth in vitro and is also 117-fold more adherent to and more invasive into Detroit 562 human epithelial cells than the encapsulated variant.Expression of six competence pathway genes and one competence-associated gene was 11 to 34-fold higher in the nonencapsulated variant than the encapsulated and transformation frequency was 3.7-fold greater.ConclusionsWe identified a new single point mutation in capsule gene cpsE of a clinical S. pneumoniae serotype 18C isolate sufficient to cause loss of capsule expression resulting in the co-existence of the encapsulated and nonencapsulated phenotype. The mutation caused phenotypic changes in growth, adherence to epithelial cells and transformability. Mutation in capsule gene cpsE may be a way for S. pneumoniae to lose its capsule and increase its colonization potential.

An unusual patient with hypercalciuria, recurrent nephrolithiasis, hypomagnesemia and G227R mutation of Paracellin-1. An unusual patient with hypercalciuria and hypomagnesemia unresponsive to thiazide diuretics

A 19-year-old female patient with hypercalciuria and recurrent nephrolithiasis/urinary tract infection unresponsive to thiazide type diuretics is presented. The patient first experienced nephrolithiasis at the age of 4 years. Afterwards, recurrent passages of stones and urinary tract infection occurred. On diagnostic evaluation at the age of 19 years, she also had hypocitraturia and hypomagnesemia. Her serum calcium concentrations were near the lower limit of normal (8.5-8.8 mg/dl; normal range: 8.5-10.5), her serum magnesium concentrations were 1.15-1.24 mg/dl (normal range: 1.4-2.5) and urinary calcium excretion was 900 mg/24 h. PTH concentrations were increased (110-156 pg/ml; normal range: 10-65). We tried to treat the patient with hydrochlorothiazide at a dose of 50 mg/day. During treatment with thiazide diuretics, PTH concentration remained high and the patient had recurrent urinary tract infections and passages of stones. Serum magnesium concentration did not normalize even under the parenteral magnesium infusion. Her mother had a history of nephrolithiasis 20 years ago. Severe hypomagnesemia in association with hypercalciuria/urinary stones is reported as a rare autosomal recessive disorder caused by impaired reabsorption of magnesium and calcium in the thick assending limp of Henle's loop. Recent studies showed that mutations in the CLDN16 gene encoding paracellin-1 cause the disorder. In exon 4, a homozygous nucleotide exchange (G679C) was identified for the patient. This results in a point mutation at position Glycine227, which is replaced by an Arginine residue (G227R). The mother was heterozygous for this mutation. G227 is located in the fourth transmembrane domain and is highly conserved in the claudin gene family. This case indicates the pathogenetic role of paracellin-1 mutation in familial hypomagnesemia with hypercalciuria and nephrocalcinosis and further underlines the risk of stone formation in heterozygous mutation carriers.

Effects of point mutations in the cytidine deaminase domains of APOBEC3B on replication and hypermutation of hepatitis B virus in vitro

APOBEC3 cytidine deaminases hypermutate hepatitis B virus (HBV) and inhibit its replication in vitro. Whether this inhibition is due to the generation of hypermutations or to an alternative mechanism is controversial. A series of APOBEC3B (A3B) point mutants was analysed in vitro for hypermutational activity on HBV DNA and for inhibitory effects on HBV replication. Point mutations inactivating the carboxy-terminal deaminase domain abolished the hypermutational activity and reduced the inhibitory activity on HBV replication to approximately 40 %. In contrast, the point mutation H66R, inactivating the amino-terminal deaminase domain, did not affect hypermutations, but reduced the inhibition activity to 63 %, whilst the mutant C97S had no effect in either assay. Thus, only the carboxy-terminal deaminase domain of A3B catalyses cytidine deaminations leading to HBV hypermutations, but induction of hypermutations is not sufficient for full inhibition of HBV replication, for which both domains of A3B must be intact.

Aromatase deficiency in a female who is compound heterozygote for two new point mutations in the P450arom gene: impact of estrogens on hypergonadotropic hypogonadism, multicystic ovaries, and bone densitometry in childhood

We report on a female who is compound heterozygote for two new point mutations in the CYP19 gene. The allele inherited from her mother presented a base pair deletion (C) occurring at P408 (CCC, exon 9), causing a frameshift that results in a nonsense codon 111 bp (37 aa) further down in the CYP19 gene. The allele inherited from her father showed a point mutation from G-->A at the splicing point (canonical GT to mutational AT) between exon and intron 3. This mutation ignores the splice site and a stop codon 3 bp downstream occurs. Aromatase deficiency was already suspected because of the marked virilization occurring prepartum in the mother, and the diagnosis was confirmed shortly after birth. Extremely low levels of serum estrogens were found in contrast to high levels of androgens. Ultrasonographic follow-up studies revealed persistently enlarged ovaries (19.5-22 mL) during early childhood (2 to 4 yr) which contained numerous large cysts up to 4.8 x 3.7 cm and normal-appearing large tertiary follicles already at the age of 2 yr. In addition, both basal and GnRH-induced FSH levels remained consistently strikingly elevated. Low-dose estradiol (E2) (0.4 mg/day) given for 50 days at the age of 3 6/12 yr resulted in normalization of serum gonadotropin levels, regression of ovarian size, and increase of whole body and lumbar spine (L1-L4) bone mineral density. The FSH concentration and ovarian size returned to pretreatment levels shortly (150 days) after cessation of E2 therapy. Therefore, we recommend that affected females be treated with low-dose E2 in amounts sufficient to result in physiological prepubertal E2 concentrations using an ultrasensitive estrogen assay. However, E2 replacement needs to be adjusted throughout childhood and puberty to ensure normal skeletal maturation and adequate adolescent growth spurt, normal accretion of bone mineral density, and, at the appropriate age, female secondary sex maturation.

Aromatase deficiency caused by a novel P450arom gene mutation: impact of absent estrogen production on serum gonadotropin concentration in a boy.

We identified a new point mutation in the CYP19 gene responsible for aromatase (P450arom) deficiency in a 46,XY male infant with unremarkable clinical findings at birth. This boy is homozygote for a 1-bp (C) deletion in exon 5 of the aromatase gene causing a frame-shift mutation. The frame-shift results in a prematurely terminated protein that is inactive due to the absence of the functional regions of the enzyme. Aromatase deficiency was suspected prenatally because of the severe virilization of the mother during the early pregnancy, and the diagnosis was confirmed shortly after birth. Four weeks after birth, the baby boy showed extremely low levels of serum estrogens, but had a normal level of serum free testosterone; in comparison with the high serum concentration of androstenedione at birth, a striking decrease occurred by 4 weeks postnatally. We previously reported elevated basal and stimulated FSH levels in a female infant with aromatase deficiency in the first year of life. In contrast, in the male infant, basal FSH and peak FSH levels after standard GnRH stimulation tests were normal. This finding suggests that the contribution of estrogen to the hypothalamic-pituitary gonadotropin-gonadal feedback mechanism is different in boys and girls during infancy and early childhood. In normal girls, serum estradiol concentrations strongly correlate with circulating inhibin levels, and thus, low inhibin levels may contribute to the striking elevation of FSH in young girls with aromatase deficiency. In contrast, estradiol levels are physiologically about a 7-fold lower in boys than in girls, and serum inhibin levels remain elevated even though levels of FSH, LH, and testosterone are decreased.

Novel mitochondrial tRNA(Ile) m.4282A>G gene mutation leads to chronic progressive external ophthalmoplegia plus phenotype

BACKGROUND/AIM To investigate the underlying pathomechanism in a 33-year-old female Caucasian patient presenting with chronic progressive external ophthalmoplegia (CPEO) plus symptoms. METHODS Histochemical analysis of skeletal muscle and biochemical measurements of individual oxidative phosphorylation (OXPHOS) complexes. Genetic analysis of mitochondrial DNA in various tissues with subsequent investigation of single muscle fibres for correlation of mutational load. RESULTS The patient's skeletal muscle showed 20% of cytochrome c oxidase-negative fibres and 8% ragged-red fibres. Genetic analysis of the mitochondrial DNA revealed a novel point mutation in the mitochondrial tRNA(Ile) (MTTI) gene at position m.4282G>A. The heteroplasmy was determined in blood, buccal cells and muscle by restriction fragment length polymorphism (RFLP) combined with a last fluorescent cycle. The total mutational load was 38% in skeletal muscle, but was not detectable in blood or buccal cells of the patient. The phenotype segregated with the mutational load as determined by analysis of single cytochrome c oxidase-negative/positive fibres by laser capture microdissection and subsequent LFC-RFLP. CONCLUSIONS We describe a novel MTTI transition mutation at nucleotide position m.4282G>A associated with a CPEO plus phenotype. The novel variant at position m.4282G>A disrupts the middle bond of the D-stem of the tRNA(Ile) and is highly conserved. The conservation and phenotype-genotype segregation strongly suggest pathogenicity and is in good agreement with the MTTI gene being frequently associated with CPEO. This novel variant broadens the spectrum of MTTI mutations causing CPEO.

A residue close to ?1 loop F disrupts modulation of GABAA receptors by benzodiazepines while their binding is maintained

Benzodiazepines act at the major isoforms of GABA type A receptors where they potentiate the current evoked by the agonist GABA. The underlying mechanism of this potentiation is poorly understood, but hypothesized to be related to the mechanism that links agonist binding to channel opening in these ligand activated ion channels. The loop F of the ?(1) and the ?(2) subunit have been implicated in channel gating, and loop F of the ?(2) subunit in the modulation by benzodiazepines. We have identified the conservative point mutation Y168F located N-terminally of loop F in the ?(1) subunit that fails to affect agonist properties. Interestingly, it disrupts modulation by benzodiazepines, but leaves high affinity binding to the benzodiazepine binding site intact. Modulation by barbiturates and neurosteroids is also unaffected. Residue ?(1) Y168 is not located either near the binding pockets for GABA, or for benzodiazepines, or close to the loop F of the ?(2) subunit. Our results support the fact, that broader regions of ligand gated receptors are conformationally affected by the binding of benzodiazepines. We infer that also broader regions could contribute to signaling from GABA agonist binding to channel opening.

Patterns of molecular response to and relapse after combination of sorafenib, idarubicin, and cytarabine in patients with FLT3 mutant acute myeloid leukemia

BACKGROUND: FMS-like tyrosine kinase 3 (FLT3) is a class III receptor tyrosine kinase involved in hematopoietic progenitor cell development. Mutations of FLT3 have been reported in about a third of patients with acute myeloid leukemia (AML), and inhibitors of FLT3 are of clinical interest. Sorafenib is an orally active multikinase inhibitor with potent activity against FLT3 and the Raf/ERK/MEK kinase pathway. METHODS: We studied the patterns of molecular response and relapse in 18 patients with mutated FLT3 treated with the combination of sorafenib, idarubicin, and cytarabine. RESULTS: The median follow-up was 9 months. Sixteen patients achieved complete remission (CR), and the other 2 patients achieved CR but lacked platelet recovery for an overall response rate of 100%. Ten patients had their FLT3-mutated clone eradicated, with 6 patients who showed some residual FLT3-mutated cells, and 2 patients who showed persistent FLT3-mutated cells. The elimination of FLT3-mutated population at the time of morphologic CR, however, was not predictive of relapse. After a median follow-up of 9 months (range, 1-16 months), 10 (55%) patients had relapsed, with a median CR duration of 8.8 months (range, 1-9.5 months). By DNA sequencing, there was no evidence of an acquired FLT3 point mutation at the time of relapse in 7 patients tested, which suggested the presence of other mechanisms of sorafenib resistance. CONCLUSION: Sorafenib, combined with chemotherapy, is effective in attaining CR, but relapses still occur.

Genetic diversity in fluoroquinolone and macrolide-resistant Campylobacter coli from pigs

The genetic diversity of 115 Campylobacter coli strains, isolated from pigs of 59 geographical distant farms in Switzerland, were characterized on the basis of their DNA fingerprints and resistance to macrolides and fluoroquinolones. Sequence analysis showed that the macrolide-resistant isolates had a point mutation in the 23S ribosomal RNA (rRNA) genes (A2075G) and that the fluoroquinolone-resistant isolates had a point mutation in the gyrase gene gyrA (C257T). One fluoroquinolone-resistant strain had an additional transition mutation in the gyrB gene (A1471C). The flaA restriction fragment length polymorphism (RFLP) genotyping revealed that 57% of the isolates were genetically different. Point mutations in the 23S rRNA and gyrA genes could be found in both genetically distant and genetically related isolates. Additionally, isolates with and without point mutations were found within individual farms and on different farms. This study showed that the ciprofloxacin and erythromycin-resistant C. coli population present on the pig farms is not issued from a common ancestral clone, but individual Campylobacter strains have most likely mutated independently to acquire resistances under the selective pressure of an antibiotic.

Impact of subunit positioning on GABAA receptor function

The major isoforms of the GABAA (gamma-aminobutyric acid type A) receptor are composed of two alpha, two beta and one gamma subunit. Thus alpha and beta subunits occur twice in the receptor pentamer. As it is well documented that different isoforms of alpha and beta subunits can co-exist in the same pentamer, the question is raised whether the relative position of a subunit isoform affects the functional properties of the receptor. We have used subunit concatenation to engineer receptors of well-defined subunit arrangement to study this question. Although all five subunits may be concatenated, we have focused on the combination of triple and dual subunit constructs. We review here what is known so far on receptors containing simultaneously alpha1 and alpha6 subunits and receptors containing beta1 and beta2 subunits. Subunit concatenation may not only be used to study receptors containing two different subunit isoforms, but also to introduce a point mutation into a defined position in receptors containing either two alpha or beta subunits, or to study the receptor architecture of receptors containing unconventional GABAA receptor subunits. Similar approaches may be used to characterize other members of the pentameric ligand-gated ion channel family, including nicotinic acetylcholine receptors, glycine receptors and 5-HT3 (5-hydroxytryptamine) receptors.

Becker muscular dystrophy with marked divergence between clinical and molecular genetic findings: case series

Both Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused by mutations of the X-linked dystrophin gene. BMD patients are less affected clinically than DMD patients. We present five patients with a diagnosis of BMD. First, two identical twins, with a deletion of exon 48 of the dystrophin gene, who experienced prominent muscle cramps from the age of three. The histopathological examination of muscle biopsies of these two twins revealed only very slight muscle fiber alterations. Second, two brothers who displayed marked, unusual intrafamilial variability of the clinical picture as well as showing a new point mutation in the dystrophin gene. And finally, a fifth boy who displayed a new point mutation in the dystrophin gene. Although he was clinically asymptomatic at the age of 15 and muscle biopsy only showed very minor myopathic signs, serum Creatine Kinase (CK) levels had been considerably elevated for years. Taken together, these cases add to the spectrum of marked discrepancies in clinical, histopathological and molecular genetic findings in BMD.

Homozygous Cys542-->Arg substitution in GPIIIa in a Swiss patient with type I Glanzmann's thrombasthenia

Glanzmann's thrombasthenia (GT) arises from a qualitative or quantitative defect in the GPIIb-IIIa complex (integrin alphaIIbbeta3), the mediator of platelet aggregation. We describe a patient in whom clinical and laboratory findings typical of type I GT were found together with a second pathology involving neurological and other complications symptomatic of tuberous sclerosis. Analysis of platelet proteins by Western blotting revealed trace amounts of normally migrating GPIIb and equally small amounts of GPIIIa of slightly slower than normal migration. Flow cytometry confirmed a much decreased binding to platelets of monoclonal antibodies to GPIIb, GPIIIa or GPIIb-IIIa, and an antibody to the alphav subunit also showed decreased binding. Nonradioactive PCR single-strand conformation polymorphism analysis followed by direct sequencing of PCR-amplified DNA fragments showed a homozygous point mutation (T to C) at nucleotide 1722 of GPIIIa cDNA and which led to a Cys542-->Arg substitution in the GPIIIa protein. The mutation gave rise to a HinP1 I restriction site in exon 11 of the GPIIIa gene and allele-specific restriction enzyme analysis of family members confirmed that a single mutated allele was inherited from each parent. This amino acid substitution presumably changes the capacity for disulphide bond formation within the cysteine-rich core region of GPIIIa and its study will provide new information on GPIIb-IIIa and alphavbeta3 structure and biosynthesis.

Large deletions in the CFTR gene: clinics and genetics in Swiss patients with CF

Cystic fibrosis (CF) is the most common life-shortening autosomal recessive disorder in Caucasians, and is associated with at least one mutation on each CF transmembrane conductance regulator (CFTR) allele. Some patients, however, with only one identifiable point mutation carry on the other allele, a large deletion that is not detected by conventional screening methods. The overall frequency of large deletions in patients with CF is estimated to be 1-3%. Using the CFTR Multiplex Ligation dependent Probe Amplification Kit (MRC-Holland, Amsterdam, Netherlands) that allows the exact detection of copy numbers from all 27 exons in the CFTR gene, we screened 50 patients with only one identified mutation for large deletions in the CFTR gene. Each detected deletion was confirmed using our real-time polymerase chain reaction (PCR) assay and deletion-specific PCR reactions using junction fragment primers. We detected large deletions in eight patients (16%). These eight CF alleles belong to four different deletion types (CFTRindel2, CFTRdele14b-17b, CFTRdele17a-17b and CFTRdele 2-9) whereof the last is novel. Comparing detailed clinical data of all these patients with CF and the molecular genetic findings, we were able to elaborate criteria for deletion screenings and possible genotype-phenotype associations. In conclusion, we agree with other authors that deletion screenings should be implemented in routine genetic diagnostics of CF.