Molecular analysis of the cystinuria disease gene: identification of four new mutations,one large deletion,and one polymorphism

A cystinuria disease gene (rBAT) has recently been identified, but evidence strongly suggests that only Type-I cystinuria is due to mutations in this gene. Sixteen point mutations and a large deletion causing the disease have so far been described in the rBAT gene sequence. To identify new mutated alleles, genomic DNA was analyzed, after the determination of the entire genomic structure of the rBAT gene, by RNA-single strand conformation polymorphism analysis, an accurate and sensitive method able to detect nucleotide changes. Four new point mutations, a large deletion, and a common intragenic polymorphism were detected. These new mutations increase to 22 the number of mutated alleles so far characterized in rBAT. In addition, the frequency of 21 mutations was assessed in a sample of accurately defined Type-I cystinuria choromosomes. They account for about 58% of all Type-I chromosomes, mutation M467T being the most common (0.26). Received: 15 March 1996 / Revised: 17 May 1996     

Isolation and characterization of phi X174 mutants carrying lethal missense mutations in gene G.

A previously constructed Escherichia coli transformant carrying a functional copy of bacteriophage phi X174 gene G on a plasmid, p phi XG, was used to isolate gene G mutants carrying temperature sensitive and lethal missense mutations. Two of the mutations have been characterized by sequencing: one carries a G --> A transition at residue 2821 producing a Gly --> Ser change in codon 143 of the G spike protein; the other carries an A --> G transition at residue 2678 producing Glu --> Gly change in codon 95. Sequencing DNA from 2 other mutants carrying lethal mutations that are rescued with p phi XG did not reveal any changes in the coding sequence. The lesion is believed to be in the intercistronic region between genes F and G. The adsorption kinetics for these mutants appear to be normal. Their burst size is about 25% that of wild type phi X174 on the host carrying p phi XG. These results along with previous results from the senior author's laboratory demonstrate that p phi XG can be used to rescue any gene G mutant of phi X174 regardless of the nature of the mutation involved.     

Molecular pathogenesis of sialic acid storage diseases: insight gained from four missense mutations and a putative polymorphism of human sialin

Background information. Free sialic acid storage diseases are caused by mutations of a lysosomal sialic acid transporter called sialin. We showed recently that the milder clinical form, Salla disease, and a related non‐Finish case, are characterized by residual transport, whereas sialin mutants found in lethal infantile cases are inactive. In the present study, we have characterized the molecular effects of a putative polymorphism (M316I) and of four pathogenic mutations associated with either infantile (G127E and R57C) or Salla‐like (G409E) phenotypes, or both (G328E). The transport activity of human sialin was analysed using a novel assay that was based on a construct without the functional lysosomal sorting motif, which is expressed at the plasma membrane. Results. The lysosomal localization of human sialin was not (M316I and G328E) or only partially (R57C, G127E and G409E) affected by the missense mutations. In contrast, all pathogenic mutations abolished transport, whereas the putative M316I polymorphism induced an approx. 5‐fold decrease of sialic acid transport. Conclusions. The molecular effects of the R57C and G127E mutations strengthen the conclusion that the infantile phenotype is caused by loss‐of‐function mutations. On the other hand, the milder severity of the heterozygous G409E patient may reflect an incomplete expression of the splicing mutation present on the second allele. In the case of the G328E mutation, found in the homozygous state in a clinically heterogeneous family, the apparent severity of the transport phenotype suggests that the genetic or environmental factors underlying this clinical heterogeneity might be protective.     

Mutation update of spinal muscular atrophy in Spain: molecular characterization of 745 unrelated patients and identification of four novel mutations in the SMN1 gene

Spinal muscular atrophy (SMA) is caused by mutations in the SMN1 gene. We have studied the molecular pathology of SMA in 745 unrelated Spanish patients using PCR-RFLP, SMN gene dosage analysis, linkage studies, long-range PCR and direct sequencing. Our systematic approach allowed us to complete genetic testing and risk assessment in 736 SMA patients (98.8%). Females were more frequently affected by the acute form of the disease (type I), whereas chronic forms (type II–III) predominated in males (p < 0.008). Absence of the SMN1 gene was detected in 671 patients (90%), and hybrid SMN1–SMN2 genes were observed in 37 cases (5%). Furthermore, we detected 13 small mutations in 28 patients (3.8%), four of which were previously identified in other populations (c.91dupT; c.770_780dup11; p.Tyr272Cys and p.Thr274Ile), while five mutations were found to date only in Spanish patients (c.399_402delAGAG, p.Ile116Phe, p.Gln136Glu, c.740dupC and c.834+2T>G). The c.399_402delAGAG mutation accounted for 1.9% of all Spanish SMA patients. Finally, we discovered four novel mutations: c.312dupA, c.411delT, p.Trp190X and p.Met263Thr. Our results confirm that most SMA cases are due to large genetic rearrangements in the repetitive region of the SMA locus, resulting in absence-dysfunction of the SMN1 gene. By contrast, ancestrally inherited small mutations are responsible for only a small number of cases. Four prevalent changes in exons 3 and 6 (c.399_402delAGAG; c.770_780dup11; p.Tyr272Cys; p.Thr274Ile) accounted for almost 70% of our patients with these subtle mutations. An SMN–SMN dimer model featuring tight hydrophobic-aromatic interactions is proposed to explain the impact of mutations at the C-terminal end of the protein.     

Molecular characterization of loss-of-function mutations in PCSK9 and identification of a compound heterozygote

Elevated levels of circulating low-density lipoprotein cholesterol (LDL-C) play a central role in the development of atherosclerosis. Mutations in proprotein convertase subtilisin/kexin type 9 (PCSK9) that are associated with lower plasma levels of LDL-C confer protection from coronary heart disease. Here, we show that four severe loss-of-function mutations prevent the secretion of PCSK9 by disrupting synthesis or trafficking of the protein. In contrast to recombinant wild-type PCSK9, which was secreted from cells into the medium within 2 hours, the severe loss-of-function mutations in PCSK9 largely abolished PCSK9 secretion. This finding predicted that circulating levels of PCSK9 would be lower in individuals with the loss-of-function mutations. Immunoprecipitation and immunoblotting of plasma for PCSK9 provided direct evidence that the serine protease is present in the circulation and identified the first known individual who has no immunodetectable circulating PCSK9. This healthy, fertile college graduate, who was a compound heterozygote for two inactivating mutations in PCSK9, had a strikingly low plasma level of LDL-C (14 mg/dL). The very low plasma level of LDL-C and apparent good health of this individual demonstrate that PCSK9 plays a major role in determining plasma levels of LDL-C and provides an attractive target for LDL-lowering therapy.     

Molecular heterogeneity of acute intermittent porphyria: identification of four additional mutations resulting in the CRIM-negative subtype of the disease.

Four mutations of the porphobilinogen (PBG) deaminase gene that result in cross-reacting immunological material (CRIM)-negative forms of acute intermittent porphyria (AIP) have been identified by in vitro amplification of cDNA from patients and by cloning of the amplified products in a bacterial expression vector. One mutation is a single base deletion which causes a frameshift and which is expected to result in the synthesis of a truncated protein. Two other mutations consist of single base substitutions and lead to amino acid changes. The fourth mutation is a single base substitution producing an aberrant splicing and resulting in an mRNA which would encode a protein missing three amino acids. DNAs from 16 unrelated CRIM-negative AIP patients were screened for the presence of these four mutations, by hybridization with oligonucleotides specific for each of the mutations, but none of the four mutations was identified in additional patients. The results indicate that mutations responsible for CRIM-negative AIP are highly heterogenous.     

Molecular characterization of minor gene rearrangements in Finnish patients with heterozygous familial hypercholesterolemia: identification of two common missense mutations (Gly823-->Asp and Leu380-->His) and eight rare mutations of the LDL receptor gene.

Two deletions of the low-density lipoprotein (LDL) receptor gene were previously shown to account for about two thirds of all mutations causing familial hypercholesterolemia (FH) in Finland. We screened the DNA samples from a cohort representing the remaining 30% of Finnish heterozygous FH patients by amplifying all the 18 exons of the receptor gene by PCR and searching for DNA variations with the SSCP technique. Ten novel mutations were identified, comprising two nonsense and seven missense mutations as well as one frameshift mutation caused by a 13-bp deletion. A single nucleotide change, substituting adenine for guanidine at position 2533 and resulting in an amino acid change of glycine to aspartic acid at codon 823, was found in DNA samples from 14 unrelated FH probands. This mutation (FH-Turku) affects the sequence encoding the putative basolateral sorting signal of the LDL receptor protein; however, the exact functional consequences of this mutation are yet to be examined. The FH-Turku gene and another point mutation (Leu380-->His or FH-Pori) together account for approximately 8% of the FH-causing genes in Finland and are particularly common among FH patients from the southwestern part of the country (combined, 30%). Primer-introduced restriction analysis was applied for convenient assay of the FH-Turku and FH-Pori point mutations. In conclusion, this paper demonstrates the unique genetic background of FH in Finland and describes a commonly occurring FH gene with a missense mutation closest to the C terminus thus far reported.     

Molecular screening of Batten disease: identification of a missense mutation (E295K) in the CLN3 gene

Batten disease, the juvenile form of neuronal ceroid lipofuscinosis, is a prevalent neuron degenerative disorder of childhood. A 1.02-kb genomic deletion in the Batten disease gene CLN3 has been determined to be a common mutation. We developed a PCR method to screen for this deletion and tested 43 Batten disease probands. We found 36% (31/86) of Batten disease chromosomes did not carry the 1.02-kb deletion. Of the three heterozygotes for the 1.02-kb deletion, a novel G-to-A missense mutation at nucleotide 1020 of the CLN3 cDNA sequence was found on two of the non-1.02-kb deletion chromosomes. The missense mutation resulted in a substitution of glutamic acid (E) by lysine (K) at position 295 (E295 K). The E295 K mutation causes a change in predicted local protein conformation. This glutamic acid is a highly conserved acidic amino acid, being present in human, mouse, dog and yeast, which suggests it may play an important role in the function of the Batten disease protein. Received: 12 May 1997 / Accepted: 21 August 1997     

Molecular characterization of the first missense mutation in the fibrinogen Aalpha-chain gene identified in a compound heterozygous afibrinogenemic patient

Congenital afibrinogenemia is a rare coagulopathy characterized by extremely low levels of functional and immunoreactive fibrinogen in plasma, associated with a hemorrhagic phenotype of variable severity. It is transmitted as an autosomal recessive trait and is invariantly associated with mutations affecting 1 of the 3 fibrinogen genes (FGA, FGB, and FGG, coding for Aalpha, Bbeta, and gamma chain, respectively). Most genetic defects causing afibrinogenemia are truncating mutations, whereas only few missense mutations (6) have been identified so far, all located in FGB. In this study, the mutational screening of an afibrinogenemic Italian male identified the first missense mutation (Met51Arg) in FGA leading to afibrinogenemia. The patient was a compound heterozygote for a previously described frameshift mutation (1215delT) in the same gene. Met51Arg involves a residue located at the very beginning of the coiled-coil domain, in a region demonstrated to play a pivotal role in hexamer formation. In-vitro expression experiments showed that Met51Arg strongly reduces secretion of hexameric fibrinogen, whereas traces of not completely assembled trimeric intermediate were found in conditioned media. Western blot analysis on the proband's plasma confirmed the presence in vivo of the trimeric fibrinogen, supporting the hypothesis that Met51Arg prevents the final step of fibrinogen assembly.     

Sequence and structural characterization of tbnat gene in isoniazid-resistant Mycobacterium tuberculosis: identification of new mutations

One of the key issues of current radiation research is the biological effect of low doses. Unfortunately, low dose science is hampered by the unavailability of easily performable, reliable and sensitive quantitative biomarkers suitable detecting low frequency alterations in irradiated cells. We applied a quantitative real time polymerase chain reaction (qRT-PCR) based protocol detecting common deletions (CD) in the mitochondrial genome to assess direct and non-targeted effects of radiation in human fibroblasts. In directly irradiated (IR) cells CD increased with dose and was higher in radiosensitive cells. Investigating conditioned medium-mediated bystander effects we demonstrated that low and high (0.1 and 2Gy) doses induced similar levels of bystander responses and found individual differences in human fibroblasts. The bystander response was not related to the radiosensitivity of the cells. The importance of signal sending donor and signal receiving target cells was investigated by placing conditioned medium from a bystander response positive cell line (F11-hTERT) to bystander negative cells (S1-hTERT) and vice versa. The data indicated that signal sending cells are more important in the medium-mediated bystander effect than recipients. Finally, we followed long term effects in immortalized radiation sensitive (S1-hTERT) and normal (F11-hTERT) fibroblasts up to 63 days after IR. In F11-hTERT cells CD level was increased until 35 days after IR then reduced back to control level by day 49. In S1-hTERT cells the increased CD level was also normalized by day 42, however a second wave of increased CD incidence appeared by day 49 which was maintained up to day 63 after IR. This second CD wave might be the indication of radiation-induced instability in the mitochondrial genome of S1-hTERT cells. The data demonstrated that measuring CD in mtDNA by qRT-PCR is a reliable and sensitive biomarker to estimate radiation-induced direct and non-targeted effects.     

Novel missense MTTP gene mutations causing abetalipoproteinemia

Objective

The microsomal triglyceride transfer protein (MTTP) plays a critical role in the formation of hepatic very low density lipoprotein. Abetalipoproteinemia (ABL) is a rare, naturally occurring extreme form of MTTP inhibition, which is characterized by the virtual absence of apolipoprotein (apo) B-containing lipoproteins in blood. The goal of this study was to examine the effect that four novel MTTP missense mutations had on protein interactions, expression and lipid-transfer activity, and to determine which mutations were responsible for the ABL phenotype observed in two patients.

Approach and results

In two patients with ABL, we identified in MTTP a novel frameshift mutation (K35Ffs*37), and four novel missense mutations, namely, G264R, Y528H, R540C, and N649S. When transiently expressed in COS-7 cells, all missense MTTP mutations interacted with apoB17, apoB48, and protein disulfide isomerase. Mutations Y528H and R540C, however, displayed negligible levels of MTTP activity and N649S displayed a partial reduction relative to the wild-type MTTP. In contrast, G264R retained full lipid-transfer activity.

Conclusions

These studies indicate that missense mutations Y528H, R540C, and N649S appear to cause ABL by reducing MTTP activity rather than by reducing binding of MTTP with protein disulfide isomerase or apoB. The region of MTTP containing amino acids 528 and 540 constitutes a critical domain for its lipid-transfer activity.     

Detection of two missense mutations and characterization of a repeat polymorphism in the factor VII gene (F7)

Summary The 3 portion of the coagulation factor VII gene, containing the activation and serine protease domains, was investigated in four subjects with factor VII deficiency by temperature gradient gel electrophoresis and sequencing of polymerase chain reaction (PCR) products. Molecules displaying an altered melting behaviour were detected in three subjects, and direct sequencing showed two mutations. A G-to-T transversion causing a missense mutation, Cys-310 to Phe, suppresses a disulphide bond conserved in the catalytic domain of all serine proteases. This mutation, which in the homozygous form causes a severe reduction in protease activity (4%), was found in two patients from different Italian regions. A G-to-A transition, which gives rise to a missense mutation, Arg-304 to Gln, and is associated with the factor VII Padua variant, was found in the heterozygous form in a subject also affected by von Willebrand disease. Two polymorphic alleles, which differ in one repeat monomer element, were precisely mapped in a region spanning the exon-intron 7 border of the factor VII gene and studied in families with factor VII or X deficiency.     

Molecular cloning of chicken hepatic histidase and the regulation of histidase mRNA expression by dietary protein

Chicken hepatic histidase activity varies with dietary protein consumption, but the mechanisms responsible for this alteration in activity are unclear. In the present research, the complete coding sequence and deduced amino acid sequence for chicken histidase was determined from clones isolated from a chicken liver cDNA library. The deduced amino acid sequence of chicken histidase has greater than 85% identity with the amino acid sequences of rat, mouse, and human histidase. In a series of four experiments, broiler chicks were allowed free access for 1.5, 3, 6, or 24 h to a low (13 g/100 g diet), basal (22 g/100 g diet) and high (40 g/100 g diet) protein diet. In the final experiment 5, chicks were allowed free access for 24 h to the basal, high protein diet or the basal diet supplemented with three different levels of l-histidine (0.22 g/100 g diet, 0.43 g/100 g diet or 0.86 g/100 g diet). There were no differences in the expression of the mRNA for histidase at 1.5 h, but at 3 h, histidase mRNA expression was significantly (P < .05) greater in chicks fed the high protein diet compared to chicks fed the low protein diet. At 6 and 24 h, histidase mRNA expression was significantly enhanced in chicks fed the high protein diet, and significantly reduced in chicks fed the low protein diet, compared with chicks fed the basal diet. Histidase mRNA expression was not altered by supplementing the basal diet with histidine. The results suggest that previously observed alterations in the activity of histidase, which were correlated to dietary protein intake, are mediated by rapid changes in the mRNA expression of this enzyme, and are not necessarily related to dietary histidine intake.     

Functional characterization of pathogenic human MSH2 missense mutations in Saccharomyces cerevisiae

Hereditary nonpolyposis colorectal cancer (HNPCC) is associated with defects in DNA mismatch repair. Mutations in either hMSH2 or hMLH1 underlie the majority of HNPCC cases. Approximately 25% of annotated hMSH2 disease alleles are missense mutations, resulting in a single change out of 934 amino acids. We engineered 54 missense mutations in the cognate positions in yeast MSH2 and tested for function. Of the human alleles, 55% conferred strong defects, 8% displayed intermediate defects, and 38% showed no defects in mismatch repair assays. Fifty percent of the defective alleles resulted in decreased steady-state levels of the variant Msh2 protein, and 49% of the Msh2 variants lost crucial protein-protein interactions. Finally, nine positions are predicted to influence the mismatch recognition complex ATPase activity. In summary, the missense mutations leading to loss of mismatch repair defined important structure-function relationships and the molecular analysis revealed the nature of the deficiency for Msh2 variants expressed in the tumors. Of medical relevance are 15 human alleles annotated as pathogenic in public databases that conferred no obvious defects in mismatch repair assays. This analysis underscores the importance of functional characterization of missense alleles to ensure that they are the causative factor for disease.     

Genome-wide identification and characterization of HSP70 gene family in four species of cotton

Heat shock proteins (HSPs) are important elements of the cellular group of molecular chaperones. Specifically, HSP70 proteins protect cells from being damaged when plants are exposed to environmental stresses. These proteins are catalysts that manage the correct folding of other proteins, and they play a key role in the development of tolerance against biotic and abiotic stresses. In the present study, 113 HSP70 genes were retrieved from the available genome assemblies of four cotton species, including Gossypium hirsutum, G. barbadense, G. arboreum, and G. raimondii. The HSP70 genes were clustered into 11 subfamilies based on phylogeny. One hundred and nine (109) gene duplications were found across these four species. Localization of genes revealed that several HSP70 genes reside in the cytoplasm. Synonymous and non-synonymous substitution rates revealed that functional segregation of HSP70 genes in cotton is due to purifying selection. Furthermore, HSP70 genes in cotton are expressed constitutively during developmental stages. These findings are valuable to understand the complex mechanism of HSP70 gene regulation that occurs in signaling pathways in response to plant stress.