ApoB gene nonsense and splicing mutations in a compound heterozygote for familial hypobetalipoproteinemia.

Two novel apoB gene mutations were identified in a patient (CM) with phenotypic homozygous hypobetalipoproteinemia. Haplotype analysis of the apoB alleles from this patient and his family members revealed him to be a genetic compound for the disease. In contrast to previous studies of other hypobetalipoproteinemic patients, no clues existed as to where in the apoB gene the molecular defects resided. Therefore, it was necessary to characterize the apoB genes of the patient by sequence analysis. The apoB gene contains 29 exons and is 43 kb in length. The gene encodes a 14.1 kb mRNA and a 4563 amino acid protein. Both apoB alleles from the patient were cloned via 26 sets of polymerase chain reactions (PCR). These clones contained a total of approximately 24 kb of apoB gene sequence, including regions 5' and 3' to the coding region, 29 exons, and the intron/exon junctions. Complete DNA sequence analysis of these clones showed that each apoB allele had a mutation. In the paternal apoB allele, there was a splicing mutation. The first base of the dinucleotide consensus sequence (GT) in the 5' splice donor site in intron 5 was replaced by a T. It is likely that this base substitution interferes with proper splicing and results in the observed absence of plasma apoB. In the maternal apoB allele, there was a nonsense mutation. The first base of the Arg codon (CGA) at residue 412 in exon 10 was replaced by a T, resulting in a termination codon (TGA). The nonsense mutation is likely to terminate translation after residue 411 resulting in a severely truncated protein only 9% of the length of B-100.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of a splice-site mutation in the aldolase B gene from an individual with hereditary fructose intolerance.

Hereditary fructose intolerance (HFI) is a potentially fatal autosomal recessive disease of carbohydrate metabolism. HFI patients exhibit a deficiency of fructose 1-phosphate aldolase (aldolase B), the isozyme expressed in tissues that metabolize fructose. The eight protein-coding exons, including splicing signals, of the aldolase B gene from one HFI patient were amplified by PCR. Dot-blot hybridization of the amplified DNA with allele-specific oligonucleotide (ASO) probes revealed a previously described A149P mutation in one allele from the proband. The mutation in the other allele was identified by direct sequencing of the double-stranded PCR-amplified material from the proband. The nucleotide sequence of exon 9 revealed a 7-base deletion/1-base insertion (delta 7 + 1) at the 3' splice site of intron 8 in one allele. This mutation was confirmed by cloning PCR-amplified exon 9 of the proband and determining the sequence of each allele separately. ASO analysis of 18 family members confirmed the Mendelian inheritance of both mutant alleles. The implications of this unique splice-site mutation in HFI are discussed.     

Mutations in the lysosomal beta-galactosidase gene that cause the adult form of GM1 gangliosidosis.

Three adult patients with acid beta-galactosidase deficiency/GM1 gangliosidosis who were from two unrelated families of Scandinavian descent were found to share a common point mutation in the coding region of the corresponding gene. The patients share common clinical features, including early dysarthria, mild ataxia, and bone abnormalities. When cDNA from the two patients in family 1 was PCR amplified and sequenced, most (39/41) of the clones showed a C-to-T transition (C-->T) at nucleotide 245 (counting from the initiation codon). This mutation changes the codon for Thr(ACG) to Met(ATG). Mutant and normal sequences were also found in that position in genomic DNA, indicating the presence of another mutant allele. Genomic DNA from the patient in family 2 revealed the same point mutation in one allele. It was determined that in each family only the father carried the C-->T mutation. Expression studies showed that this mutation produced 3%-4% of beta-galactosidase activity, confirming its deleterious effects. The cDNA clones from the patients in family 1 that did not contain the C-->T revealed a 20-bp insertion of intronic sequence between nucleotides 75 and 76, the location of the first intron. Further analysis showed the insertion of a T near the 5' splice donor site which led to the use of a cryptic splice site. It appears that the C-->T mutation results in enough functional enzyme to produce a mild adult form of the disease, even in the presence of a second mutation that likely produces nonfunctional enzyme.     

Characterization of the normal alpha 1-antitrypsin allele Vmunich: a variant associated with a unique protein isoelectric focusing pattern.

alpha 1-Antitrypsin (alpha 1AT), the major serum inhibitor of neutrophil elastase, is a highly polymorphic protein associated with isoelectric focusing (IEF) patterns typical for each variant. alpha 1AT Vmunich, a previously unreported normal alpha 1AT variant, has a unique IEF banding pattern in which the 7 and 8 alpha 1AT protein bands focus with the normal M-type 7 and 8 bands, despite the fact that the major fraction of the Vmunich protein focuses in the "V" region of the IEF gel. To characterize the molecular basis of this variant and its unique IEF pattern, DNA sequence analysis of the coding exons of the Vmunich alpha 1AT gene was carried out using the polymerase chain reaction. The Vmunich allele differed from the common normal M1(Val213) alpha 1AT allele by a single nucleotide substitution of cytosine for adenosine, with the resultant amino acid change Asp2 GAT----Ala GCT. Inheritance of the allele was confirmed by family analysis using allele-specific amplification with the polymerase chain reaction. The Asp2----Ala mutation explains the cathodal position of the Vmunich protein on IEF, as there is a substitution of a negatively charged amino acid by a neutral one.(ABSTRACT TRUNCATED AT 250 WORDS)     

Homologous recombination among three intragene Alu sequences causes an inversion-deletion resulting in the hereditary bleeding disorder Glanzmann thrombasthenia.

The crucial role of the human platelet fibrinogen receptor in maintaining normal hemostasis is best exemplified by the autosomal recessive bleeding disorder Glanzmann thrombasthenia (GT). The platelet fibrinogen receptor is a heterodimer composed of glycoproteins IIb (GPIIb) and IIIa (GPIIIa). Platelets from patients with GT have a quantitative or qualitative abnormality in GPIIb and GPIIIa and can neither bind fibrinogen nor aggregate. Very few genetic defects have been identified that cause this disorder. We describe a kindred with GT in which the affected individuals have a unique inversion-deletion mutation in the gene for GPIIIa. Patient platelets lacked both GPIIIa protein and mRNA. Southern blots of patient genomic DNA probed with an internal 1.0-kb GPIIIa cDNA suggested a large rearrangement of this gene but were normal when probed with small GPIIIa cDNA fragments that were outside the mutation. Cytogenetics and pulsed-field gel analysis of the GPIIIa gene were normal, making a translocation or a very large rearrangement unlikely. Additional Southern analyses suggested that the abnormality was not a small insertion. We constructed a patient genomic DNA library and isolated fragments containing the 5' and 3' breakpoints of the mutation. The nucleotide sequence from these genomic clones was determined and revealed that, relative to the normal gene, the mutant allele contained a 1-kb deletion immediately preceding a 15-kb inversion. The DNA breaks occurred in two inverted and one forward Alu sequence within the gene for GPIIIa and in the left, right, and left arms, respectively, of these sequences. There was a 5-bp repeat at the 3' terminus of the inversion.(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel mutation in the invariant AG of the acceptor splice site of intron 4 of the beta-hexosaminidase alpha-subunit gene in two unrelated American black GM2-gangliosidosis (Tay-Sachs disease) patients.

Samples of genomic DNA from three unrelated American black infants having both biochemical and clinical features of classical infantile Tay-Sachs disease were sequenced following PCR amplification. A G----T transversion was observed in the AG acceptor splice site preceding exon 5 of the beta-hexosaminidase alpha-subunit gene in the first black family. This transversion changed the acceptor splice site from the consensus sequence, AG, to AT, thereby interfering with splicing at this intron 4/exon 5 junction. The proband was homozygous for this mutation; his mother and a brother are heterozygous. The same mutation was found in a second, apparently unrelated, black GM2-gangliosidosis patient. The second patient was a compound heterozygote, as only one allele carried this mutation. The mother and a brother in this second family are carriers for this mutation, while the father and a noncarrier sister are normal for this region of the gene. The third proband did not have this mutation; nor did the mother of a fourth black proband. Eight other independently ascertained non-black, non-Jewish, GM2-gangliosidosis families did not have this mutation. The observation of the same novel mutation in two unrelated black GM2-gangliosidosis patients indicates that the American black population has segregating within it at least one GM2-gangliosidosis mutation which may be specific to this population and not a result of migration.     

一个46,XY"女性"不育症家系的遗传学分析

运用常规的染色体G带分析和基因分析技术对-46,XY男性女性化家系进行遗传学分析,发现：先证者及其妹妹的染色体核型为46,XY,其母亲和父亲的核型正常;对睾丸决定基因（SRY）和雄激素受体基因（AR）进行突变检测,在SRY基因的整个编码区中没有发现突变,而AR基因的第7个外显子的第840个密码子由CGT（编码精氨酸）变为CAT（编码组氨酸）,这一改变可能是导致核型为46,XY女性化而发生不育。     

Two new arylsulfatase A (ARSA) mutations in a juvenile metachromatic leukodystrophy (MLD) patient.

Fragments of the arylsulfatase A (ARSA) gene from a patient with juvenile-onset metachromatic leukodystrophy (MLD) were amplified by PCR and ligated into MP13 cloning vectors. Clones hybridizing with cDNA for human ARSA were selected, examined for appropriate size inserts, and used to prepare single-stranded phage DNA. Examination of the entire coding and most of the intronic sequence revealed two putative disease-related mutations. One, a point mutation in exon 3, resulted in the substitution of isoleucine by serine. Introduction of this alteration into the normal ARSA cDNA sequence resulted in a substantial decrease in ARSA activity on transient expression in cultured baby hamster kidney cells. About 5% of the control expression was observed, suggesting a small residual activity in the mutated ARSA. The second mutation, a G-to-A transition, occurred in the other allele and resulted in an altered splice-recognition sequence between exon 7 and the following intron. The mutation also resulted in the loss of a restriction site. Apparently normal levels of mRNA were generated from this allele, but no ARSA activity or immuno-cross-reactive material could be detected. A collection of DNA samples from known or suspected MLD patients, members of their families, and normal controls was screened for these mutations. Four additional individuals carrying each of the mutations were found among the nearly 100 MLD patients in the sample. Gene segregation in the original patient's family was consistent with available clinical and biochemical data. No individuals homozygous for either of these two mutations were identified. However, combinations with other MLD mutations suggest that the point mutation in exon 3 does result in some residual enzyme activity and is associated with late-onset forms of the disease. The splice-site mutation following exon 7 produces late-infantile MLD when combined with other enzyme-null mutations, implying that it is completely silent enzymatically.     

Reduced mRNA and a nonsense mutation in the insulin-receptor gene produce heritable severe insulin resistance.

Leprechaunism is an autosomal recessive syndrome of severe insulin resistance and is characterized by intrauterine growth restriction, acanthosis nigricans, hirsutism, and loss of glucose homeostasis. Here we report a new female patient of Hispanic and Afro-American descent whose fibroblasts and lymphoblasts had markedly impaired insulin binding (less than 10% of that in controls). Insulin binding to lymphoblasts established from both unrelated parents was partially impaired. Insulin-like growth factor-I (IGF-I) and epidermal growth factor (EGF) binding to the patient's fibroblasts were within the normal range. Insulin stimulation of receptor autophosphorylation and kinase activity was markedly reduced in the patient's fibroblasts. The patient's fibroblasts had both a reduced number of immunoreactive insulin receptor (6% of those in controls) and concomitantly reduced amounts of insulin-receptor mRNA, suggesting that both mutations inherited by the patient reduced insulin-receptor mRNA. Sequencing of the insulin-receptor gene and cDNA indicated that the patient was heterozygous for a paternally derived mutation at bp 1333, converting Arg372 to a STOP codon. This nonsense mutation was observed in the insulin-receptor gene, but not in cDNA, indicating reduced amounts of mRNA for the allele containing this mutation. The coding sequence of the maternally inherited insulin-receptor allele was normal. Both the marked reduction in insulin-receptor mRNA in the compound heterozygous fibroblasts of the proband and the partially reduced insulin binding in maternal cells suggest that the maternally derived mutation is located in an insulin-receptor gene sequence that controls cellular mRNA content.     

Two mutant alleles of the insulin receptor gene in a family with a genetic form of insulin resistance: a 10 base pair deletion in exon 1 and a mutation substituting serine for asparagine-462

Mutations in the insulin receptor gene cause several genetic syndromes associated with extreme insulin resistance. We have studied three insulin resistant siblings with acanthosis nigricans, dental abnormalities, and acral hypertrophy. The female patient also had primary amenorrhea due to hyperandrogenism. All three patients were compound heterozygotes with two mutant alleles of the insulin receptor gene. One allele had a 10-bp deletion in the region of exon 1 encoding the hydrophobic signal peptide; this leads to a frameshift and premature chain termination at codon 61. The deletion occurs at the site of a direct repeat of a hexanucleotide sequence interrupted by a tetranucleotide sequence; the deletion may have resulted from recombination between the upstream and downstream hexanucleotide repeats. In the other mutant allele, there is a missense mutation substituting serine for Asn⁴⁶² — a mutation identified previously in one allele of the insulin receptor gene in a patient with type-A insulin resistance. The Ser⁴⁶² mutation impaired the ability of acidic pH to dissociate insulin from the receptor. Thus, like the previously described Glu⁴⁶⁰ mutation, the Ser⁴⁶² mutation may retard dissociation of insulin from the receptor in the acidic compartment of the endosome and may, as a result, accelerate the rate of receptor degradation.     

A large deletion/insertion-induced frameshift mutation of the androgen receptor gene in a family with a familial complete androgen insensitivity syndrome

Androgen insensitivity syndrome (AIS) is an X-linked recessive genetic disorder with a normal 46, XY karyotype caused by abnormality of the androgen receptor (AR) gene. One Chinese family consisting of the proband and 5 other members with complete androgen insensitivity syndrome (CAIS) was investigated. Mutation analysis by DNA sequencing on all 8 exons and flanking intron regions of the AR gene revealed a unique large deletion/insertion mutation in the family. A 287 bp deletion and 77 bp insertion (c.933_1219delins77) mutation at codon 312 resulted in a frameshift which caused a premature stop (p.Phe312Aspfs*7) of polypeptide formation. The proband's mother and grandmother were heterozygous for the mutant allele. The proband's father, uncle and grandfather have the normal allele. From the pedigree constructed from mutational analysis of the family, it is revealed that the probably pathogenic mutation comes from the maternal side.     

Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox.

Fragile X syndrome results from mutations in a (CGG)n repeat found in the coding sequence of the FMR-1 gene. Analysis of length variation in this region in normal individuals shows a range of allele sizes varying from a low of 6 to a high of 54 repeats. Premutations showing no phenotypic effect in fragile X families range in size from 52 to over 200 repeats. All alleles with greater than 52 repeats, including those identified in a normal family, are meiotically unstable with a mutation frequency of one, while 75 meioses of alleles of 46 repeats and below have shown no mutation. Premutation alleles are also mitotically unstable as mosaicism is observed. The risk of expansion during oogenesis to the full mutation associated with mental retardation increases with the number of repeats, and this variation in risk accounts for the Sherman paradox.     

Segregation of a M404V mutation of the p62/sequestosome 1 (p62/SQSTM1) gene with polyostotic Paget's disease of bone in an Italian family

Mutations of the p62/Sequestosome 1 gene (p62/SQSTM1) account for both sporadic and familial forms of Paget's disease of bone (PDB). We originally described a methionine-->valine substitution at codon 404 (M404V) of exon 8, in the ubiquitin protein-binding domain of p62/SQSTM1 gene in an Italian PDB patient. The collection of data from the patient's pedigree provided evidence for a familial form of PDB. Extension of the genetic analysis to other relatives in this family demonstrated segregation of the M404V mutation with the polyostotic PDB phenotype and provided the identification of six asymptomatic gene carriers. DNA for mutational analysis of the exon 8 coding sequence was obtained from 22 subjects, 4 PDB patients and 18 clinically unaffected members. Of the five clinically ascertained affected members of the family, four possessed the M404V mutation and exhibited the polyostotic form of PDB, except one patient with a single X-ray-assessed skeletal localization and one with a polyostotic disease who had died several years before the DNA analysis. By both reconstitution and mutational analysis of the pedigree, six unaffected subjects were shown to bear the M404V mutation, representing potential asymptomatic gene carriers whose circulating levels of alkaline phosphatase were recently assessed as still within the normal range. Taken together, these results support a genotype-phenotype correlation between the M404V mutation in the p62/SQSTM1 gene and a polyostotic form of PDB in this family. The high penetrance of the PDB trait in this family together with the study of the asymptomatic gene carriers will allow us to confirm the proposed genotype-phenotype correlation and to evaluate the potential use of mutational analysis of the p62/SQSTM1 gene in the early detection of relatives at risk for PDB.     

Complete amino acid sequence of the human alpha 5 (IV) collagen chain and identification of a single-base mutation in exon 23 converting glycine 521 in the collagenous domain to cysteine in an Alport syndrome patient.

We have generated and characterized cDNA clones providing the complete amino acid sequence of the human type IV collagen chain whose gene has been shown to be mutated in X chromosome-linked Alport syndrome. The entire translation product has 1,685 amino acid residues. There is a 26-residue signal peptide, a 1,430-residue collagenous domain starting with a 14-residue noncollagenous sequence, and a Gly-Xaa-Yaa-repeat sequence interrupted at 22 locations, and a 229-residue carboxyl-terminal noncollagenous domain. The calculated molecular weight of the mature alpha 5(IV) chain is 158,303. Analysis of genomic DNA from members of a kindred with Alport syndrome revealed a new HindIII cleavage site within the coding sequence of one of the cDNA clones characterized. The proband had a new 1.25-kilobase HindIII fragment and a lack of a 1.35-kilobase fragment, and his mildly affected female cousin had both alleles. The mutation which was located to exon 23 was sequenced from a polymerase chain reaction-amplified product, and shown to be a G----T change in the coding strand. The mutation changed the GGT codon of glycine 521 to cysteine. The same mutation was found in one allele of the female cousin. The results were confirmed by allele-specific hybridization analyses.     

A rare disease-associated mutation in the medium-chain acyl-CoA dehydrogenase (MCAD) gene changes a conserved arginine, previously shown to be functionally essential in short-chain acyl-CoA dehydrogenase (SCAD)

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is a serious and potentially fatal inherited defect in the β-oxidation of fatty acids. Approximately 80% of patients with MCAD deficiency are homozygous for a single disease-causing mutation (G985). The remaining patients (except for a few cases worldwide) are compound heterozygous with G985 in one allele. By sequencing of cloned PCR-amplified MCAD cDNA from a G985 compound heterozygous patient, we identified a C-to-T transition at position 157 as the only change in the entire coding sequence of the non-G985 allele. The presence of the T157 mutation was verified in genomic DNA from the patient and her mother by a PCR-based assay. The mutation changes a conserved arginine at position 28 (R28C) of the mature MCAD protein. The effect of the T157 mutation on MCAD protein was investigated by expression of mutant MCAD cDNA in COS-7 cells. On the basis of knowledge about the three-dimensional structure of the MCAD protein, we suggest that the mutation destroys a salt bridge between arginine²⁸ and glutamate⁸⁶, thereby affecting the formation of enzymatically active protein. Twenty-two additional families with compound heterozygous patients were tested in the PCR-based assay. The T157 mutation was identified in one of these families, which had an MCAD-deficient child who died unexpectedly in infancy. Our results indicate that the mutation is rare. It is, however, noteworthy that a homologous mutation has previously been identified in the short-chain acyl-CoA dehydrogenase (SCAD) gene of a patient with SCAD deficiency, suggesting that the conserved arginine is crucial for formation of active enzyme in the straight-chain acyl-CoA dehydrogenases.     

Genetic mosaicism of a frameshift mutation in the RET gene in a family with Hirschsprung disease

Mutations and polymorphisms in the RET gene are a major cause of Hirschsprung disease (HSCR). Theoretically, all true heterozygous patients with a new manifestation of a genetically determined disease must have parents with a genetic mosaicism of some extent. However, no genetic mosaicism has been described for the RET gene in HSCR yet. Therefore, we analyzed families with mutations in the RET gene for genetic mosaicism in the parents of the patients. Blood samples were taken from patients with HSCR and their families/parents to sequence the RET coding region. Among 125 families with HSCR, 33 families with RET mutations were analyzed. In one family, we detected a frameshift mutation due to a loss of one in a row of four cytosines in codon 117/118 of the RET gene (c.352delC) leading to a frameshift mutation in the protein (p.Leu118Cysfs*105) that affected two siblings. In the blood sample of the asymptomatic father we found a genetic mosaicism of this mutation which was confirmed in two independent samples of saliva and hair roots. Quantification of peak-heights and comparison with different mixtures of normal and mutated plasmid DNA suggested that the mutation occurred in the early morula stadium of the founder, between the 4- and 8-cell stages. We conclude that the presence of a RET mutation leading to loss of one functional allele in 20 to 25% of the cells is not sufficient to cause HSCR. The possibility of a mosaicism has to be kept in mind during genetic counseling for inherited diseases.