Heterogeneous mutations in the gene encoding the alpha-subunit of the stimulatory G protein of adenylyl cyclase in Albright hereditary osteodystrophy

Albright hereditary osteodystrophy (AHO) is an inherited disorder associated with deficient activity of the alpha-subunit of the guanine nucleotide-binding regulatory protein (Gs alpha) that couples receptors to adenylyl cyclase. To identify mutations that lead to Gs alpha deficiency, we isolated genomic DNA from patients with AHO and used the polymerase chain reaction to amplify exons of the Gs alpha genes. DNA was amplified using intron-specific oligonucleotide primers flanking exons of the Gs alpha gene. To optimize our ability to detect mutations, one oligonucleotide from each primer pair was synthesized with a 5' GC-clamp. Amplified Gs alpha gene fragments were analyzed by denaturing gradient gel electrophoresis in order to detect mutations that alter the melting point of the double-stranded DNA fragment. Using this technique, we have identified and characterized three mutations and one neutral polymorphism. The polymorphism, located in exon 5, consisted of a T-->C substitution that conserves the isoleucine residue at codon 131 (ATT-->ATC). Two mutations were missense mutations, which in one family consisted of a nucleotide substitution (T-->C) in exon 4 that results in replacement of Leu by Pro at codon 99 of the Gs alpha molecule. Affected subjects in a second family had a single base (C-->T) mutation in exon 6 that resulted in replacement of Arg by Cys at codon 165. A 4-base pair deletion (GTGG) in exon 8 at position +214 was identified in one Gs alpha allele from each affected subject in the third family. This mutation causes a frameshift after the codon for Gln213 that results in a premature stop codon 81 base pair after the deletion. Immunoblot analysis of plasma membranes prepared from cultured fibroblasts or erythrocytes indicated that levels of immunoactive Gs alpha protein were decreased in all affected subjects. We conclude that heterogeneous mutations in the gene encoding Gs alpha, including deletions and single amino acid substitutions, are responsible for Gs alpha deficiency in AHO.     

Exon scanning for mutation of the NF2 gene in schwannomas

Family studies and tumor analyses have combined to indicate that neurofibromatosis 2 (NF2), a disorder characterized by multiple benign tumors of the nervous system, and sporadic non-inherited forms of the same tumor types are both caused by inactivation of a tumor suppressor gene located in 22q12. Recently, the gene encoding merlin, a novel member of a family of cytoskeleton-associated proteins, was identified as the NF2 tumor suppressor. To facilitate the search for merlin mutations, we have defined the exon-intron boundaries for all 17 NF2 exons, including one subject to alternative splicing. We have developed polymerase chain reaction assays to amplify each exon from genomic DNA, and used these assays to perform single-strand conformation polymorphism analysis of DNA from 30 sporadic and eight NF2-derived schwannomas, the hallmark tumor type in this disorder. Of a maximum of 60 alleles scanned, 32 showed mutations affecting expression of the merlin protein. Thirty of these mutations are predicted to lead to a truncated protein due to frameshift, creation of a stop codon, or interference with normal splicing, while two are missense mutations. Thus, inactivation of merlin is a common feature underlying both inherited and sporadic forms of schwannoma.     

Complete thyroxine-binding globulin (TBG) deficiency produced by a mutation in acceptor splice site causing frameshift and early termination of translation (TBG-Kankakee)

Fourteen T4-binding globulin (TBG) variants have been identified at the gene level. They are all located in the coding region of the gene and 6 produce complete deficiency of TBG (TBG-CD). We now describe the first mutation in a noncoding region producing TBG-CD. The proband was treated for over 20 yr with L-T4 because of fatigue associated with a low concentration of serum total T4. Fifteen family members were studied showing low total T4 inherited as an X chromosome-linked trait, and affected males had undetectable TBG in serum. Sequencing of the entire coding region and promoter of the TBG gene revealed no abnormality. However, an A to G transition was found in the acceptor splice junction of intron II that produced a new HaeIII restriction site cosegregating with the TBG-CD phenotype. Sequencing exon 1 to exon 3 of TBG complementary DNA reverse transcribed from messenger RNA of skin fibroblasts from an affected male, confirmed a shift in the ag acceptor splice site. This results in the insertion of a G in exon 2 and causes a frameshift and a premature stop at codon 195. This early termination of translation predicts a truncated TBG lacking 201 amino acids.     

The Pro347Leu mutation of the rhodopsin gene in a Spanish family with autosomal dominant pigmentary retinosis

We present a Spanish family affected with autosomal dominant pigmentary retinosis in which we have identified the mutation responsible for the disease (Pro347Leu) within the rhodopsin (RHO) gene. Complete ophthalmological and electrophysiological studies were performed in 14 members of this family. The molecular study, performed by SSCP analysis of the 5 exon and the promotor region of the rhodopsin gene, direct sequentiation and restriction analysis with the enzyme Mspl, showed a C-->T change in the second base of 347 codon of RHO gene. This mutation predicts a change of proline by leucine at this position. Every patient with the mutation showed a phenotype of diffuse, early onset and severe pigmentary retinosis with a little intrafamiliar variation. The Pro347Leu mutation, that has been very frequently described among all the populations, has been identified as a cause of RP in an Spanish family.     

Two novel missense mutations in calcium-sensing receptor gene associated with neonatal severe hyperparathyroidism

Familial hypocalciuric hypercalcemia (FHH) is characterized by lifelong asymptomatic hypercalcemia without PTH hypersecretion and is inherited as an autosomal dominant trait with near 100% penetrance. In contrast, neonatal severe hyperparathyroidism (NSHPT) is a life-threatening disorder characterized by marked hypercalcemia and PTH hypersecretion. FHH/NSHPT results from inactivating mutations of the human calcium-sensing receptor (Casr) gene on chromosome 3q13.3-24. Nearly 30 different mutations of the Casr gene associated with FHH/NSHPT have been reported previously. In this report, genetic analysis of 1 Japanese NSHPT family revealed 2 novel mutations at codon 185 (CGA-->TGA/Arg-->Ter) in exon 4 of the Casr gene and at codon 670 (GGG-->GAG/Gly-->Glu) in exon 7. The Arg185Ter change was shown to occur in the proband's unaffected father and paternal grandmother as well as in the proband. The other mutation in exon 7 was shown in the proband's unaffected mother of Philippine origin as well as in the proband. This family is the first case of manifestation of more than 1 mutation in a proband's chromosomes; 1 mutation was obtained from the unaffected father, and the other was from the unaffected mother. Our observations have given us important keys to help elucidate the structure-function relationships of the Casr.     

Caudal appendage in a full-term infant

We report studies of two unrelated Japanese patients with 17alpha-hydroxylase deficiency caused by mutations of the 17alpha-hydroxylase (CYP17) gene. We amplified all eight exons of the CYP17 gene, including the exon-intron boundaries, by the polymerase chain reaction and determined their nucleotide sequences. Patient 1 had novel, compound heterozygous mutations of the CYP17 gene. One mutant allele had a guanine to thymine transversion at position +5 in the splice donor site of intron 2. This splice-site mutation caused exon 2 skipping, as shown by in vitro minigene expression analysis of an allelic construct, resulting in a frameshift and introducing a premature stop codon (TAG) 60 bp downstream from the exon 1-3 boundary. The other allele had a missense mutation of His (CAC) to Leu (CTC) at codon 373 in exon 6. These two mutations abolished the 17alpha-hydroxylase and 17,20-lyase activities. Restriction fragment length polymorphism (RFLP) analysis with a mismatch oligonucleotide showed that the patient's mother and brother carried the splice-site mutation, but not the missense mutation. Patient 2 was homozygous for a novel 1-bp deletion (cytosine) at codon 131 in exon 2. This 1-bp deletion produces a frameshift in translation and introduces a premature stop codon (TAG) proximal to the highly conserved heme iron-binding cysteine at codon 442 in microsomal cytochrome P450 steroid 17alpha-hydroxylase (P450c17). RFLP analysis showed that the mother was heterozygous for the mutation.     

Germline mutations in the 3' part of APC exon 15 do not result in truncated proteins and are associated with attenuated adenomatous polyposis coli

Familial adenomatous polyposis (FAP) is an inherited predisposition to colorectal cancer characterized by the development of numerous adenomatous polyps predominantly in the colorectal region. Germline mutations in the adenomatous polyposis coli (APC) gene are responsible for most cases of FAP. Mutations at the 5' end of APC are known to be associated with a relatively mild form of the disease, called attenuated adenomatous polyposis coli (AAPC). We identified a frameshift mutation in the 3' part of exon 15, resulting in a stop codon at 1862, in a large Dutch kindred with AAPC. Western blot analysis of lymphoblastoid cell lines derived from affected family members from this kindred, as well as from a previously reported Swiss family carrying a frameshift mutation at codon 1987 and displaying a similar attenuated phenotype, showed only the wild-type APC protein. Our study indicates that chain-terminating mutations located in the 3' part of APC do not result in detectable truncated polypeptides and we hypothesize that this is likely to be the basis for the observed AAPC phenotype.     

Evolution of precipitates in lead-implanted aluminum: A backscattering and channeling study

BACKGROUND: Thirty-six mutations that cause Gaucher disease, the most common glycolipid storage disorder, are known. Although both alleles of most patients with the disease contain one of these mutations, in a few patients one or both disease-producing alleles have remained unidentified. Identification of mutations in these patients is useful for genetic counseling. MATERIALS AND METHODS: The DNA from 23 Gaucher disease patients in whom at least one glucocerebrosidase allele did not contain any of the 36 previously described mutations has been examined by single strand conformation polymorphism (SSCP) analysis, followed by sequencing of regions in which abnormalities were detected. RESULTS: Eight previously undescribed mutations were detected. In exon 3, a deletion of a cytosine at cDNA nt 203 was found. In exon 6, three missense mutations were identified: a C-->A transversion at cDNA nt 644 (Ala176-->Asp), a C-->A transversion at cDNA nt 661 that resulted in a (Pro182-->Thr), and a G-->A transition at cDNA nt 721 (Gly202-->Arg). Two missense mutations were found in exon 7: a G-->A transition at cDNA nt 887 (Arg257-->Gln) and a C-->T at cDNA nt 970 (Arg285-->Cys). Two missense mutations were found in exon 9: a T-->G at cDNA nt 1249 (Trp378-->Gly) and a G-->A at cDNA nt 1255 (Asp380-->Asn). In addition to these disease-producing mutations, a silent C-->G transversion at cDNA nt 1431, occurring in a gene that already contained the 1226G mutation, was found in one family. CONCLUSIONS: The mutations described here and previously known can be classified as mild, severe, or lethal, on the basis of their effect on enzyme production and on clinical phenotype, and as polymorphic or sporadic, on the basis of the haplotype in which they are found. Rare mutations such as the new ones described here are sporadic in nature.     

Mutations in haemophilia A

In the present study DNA from 281 unrelated haemophilia A patients including 15 inhibitor patients has been analysed by Southern blotting technique. Using various restriction enzymes, cloned factor VIII cDNA probes and genomic fragments we have identified 14 mutations. Six of the mutations are novel partial factor VIII gene deletions. One deletion affects exon 1, two deletions concern exon 6, another deletion, of which breakpoints are sequenced, takes part of exon 16 and two deletions affect exon 26. Besides the deletions, eight point mutations have been found at the TaqI restriction sites of exons 18, 24 and 26. Five C-->T mutations resulted in nonsense mutations, one in exon 18, one in exon 26 and three in exon 24. Two G-->A mutations caused a missense mutation in exon 24 leading to an arginine/glutamine exchange. Although two patients showed this mutation, their clinical phenotypes were different, possibly due to an additional unidentified sequence polymorphism. A G-->T mutation in exon 26 substituted the arginine with leucine. All deletions and seven of the point mutations are associated with severe disease with a detectable inhibitor in the patient with the TaqI-point mutation in exon 18. One of the G-->A mutations is associated with mild haemophilia but the patient also has developed an inhibitor. Amongst these mutations the origin of the mutation could be determined in four kindred, one of which showed maternal mosaicism.     

Characterization of nine novel mutations in the CD40 ligand gene in patients with X-linked hyper IgM syndrome of various ancestry

X-linked immunodeficiency with hyper-IgM (HIGMX-1) is a rare disorder caused by defective expression of the CD40 ligand (CD40L) by activated T lymphocytes, resulting in inefficient T-B cell cooperation and failure of B cells to undergo immunoglobulin isotype switch. In the present work, we describe nine patients of various ancestry who bear different mutations in the X chromosome-specific CD40L gene. Two of the mutations were nonsense mutations, one each resulting in premature stop codons at amino acid residues 39 and 140. Three patients had single point missense mutations, one each at codons 126, 140, and 144. Another patient had a 4-bp genomic deletion in exon 2, resulting in a frameshift and premature termination. Three patients showed insertions, one each of 1, 2, and 4 nt, probably because of polymerase slippage, resulting in frameshift mutation and premature termination. Overall, these observations confirm the heterogeneity of mutations in HIGMX-1. However, the identification of two patients whose mutation involves codon 140 (previously shown to be altered in two other unrelated subjects) suggests that this may be a hotspot of mutation in HIGMX-1. In two additional patients with clinical and immunological features indistinguishable from canonical HIGMX-1, no mutation was detected in the coding sequence, in the 5' flanking region, or in the 3' UTR.     

Germ-line mutations in the neurofibromatosis 2 gene: correlations with disease severity and retinal abnormalities

Neurofibromatosis 2 (NF2) features bilateral vestibular schwannomas, other benign neural tumors, and cataracts. Patients in some families develop many tumors at an early age and have rapid clinical progression, whereas in other families, patients may not have symptoms until much later and vestibular schwannomas may be the only tumors. The NF2 gene has been cloned from chromosome 22q; most identified germ-line mutations result in a truncated protein and severe NF2. To look for additional mutations and clinical correlations, we used SSCP analysis to screen DNA from 32 unrelated patients. We identified 20 different mutations in 21 patients (66%): 10 nonsense mutations, 2 frameshifts, 7 splice-site mutations, and 1 large in-frame deletion. Clinical information on 47 patients from the 21 families included ages at onset and at diagnosis, numbers of meningiomas, spinal and skin tumors, and presence of cataracts and retinal abnormalities. We compared clinical findings in patients with nonsense or frameshift mutations to those with splice-site mutations. When each patient was considered as an independent random event, the two groups differed (P < or = .05) for nearly every variable. Patients with nonsense or frameshift mutations were younger at onset and at diagnosis and had a higher frequency and mean number of tumors, supporting the correlation between nonsense and frameshift mutations and severe NF2. When each family was considered as an independent random event, statistically significant differences between the two groups were observed only for mean ages at onset and at diagnosis. A larger data set is needed to resolve these discrepancies. We observed retinal hamartomas and/or epiretinal membranes in nine patients from five families with four different nonsense mutations. This finding, which may represent a new genotype-phenotype correlation, merits further study.     

A nucleotide polymorphism in ERCC1 in human ovarian cancer cell lines and tumor tissues

We studied the DNA sequence of the entire coding region of ERCC1 gene, in five cell lines established from human ovarian cancer (A2780, A2780/CP70, MCAS, OVCAR-3, SK-OV-3), 29 human ovarian cancer tumor tissue specimens, one human T-lymphocyte cell line (H9), and non-malignant human ovary tissue (NHO). Samples were assayed by PCR-SSCP and DNA sequence analyses. A silent mutation at codon 118 (site for restriction endonuclease MaeII) in exon 4 of the gene was detected in MCAS, OVCAR-3 and SK-OV-3 cells, and NHO. This mutation was a C-->T transition, that codes for the same amino acid: asparagine. This transition converts a common codon usage (AAC) to an infrequent codon usage (AAT), whereas frequency of use is reduced two-fold. This base change was associated with a detectable band shift on SSCP analysis. For the 29 ovarian cancer specimens, the same base change was observed in 15 tumor samples and was associated with the same band shift in exon 4. Cells and tumor tissue specimens that did not contain the C-->T transition, did not show the band shift in exon 4. Our data suggest that this alteration at codon 118 within the ERCC1 gene, may exist in platinum-sensitive and platinum-resistant ovarian cancer tissues.     

Sequence and expression of chicken beta A2- and beta B3-crystallins

Two novel point mutations have been identified in the low density lipoprotein receptor (LDLR) gene of a South African Indian patient with a clinical diagnosis of homozygous familial hypercholesterolemia (FH). The patient is a compound heterozygote, whose paternally-inherited allele has a single base substitution of A to T at position + 1. This conversion of the initiation codon ATG (methionine) to TTG (leucine) would abolish initiation of translation at the normal site, and consequently the synthesis of any normal LDLR molecules. The second mutation identified is a C to A base change at nucleotide position 1176 in exon 8, which creates a stop codon at cysteine-371. Except for previously-described polymorphisms in specific regions of the LDLR gene, the mutations identified in exons 1 and 8 were the only variants observed by screening enzymatically amplified genomic DNA comprising the entire coding and promoter region of the LDLR gene by combined heteroduplex-single-strand conformation polymorphism analysis and by direct sequencing. Cultured cells from the proband expressed no functional LDLR activity and contained no receptor protein that could be detected by antibody binding. These findings are consistent with the nature of the two base changes identified and provide evidence that the mutations cause FH in the proband and his affected family members. The mutations, designated M-21L and C371X, were absent in 17 apparently unrelated Indian hypercholesterolemics and 200 normal chromosomes screened.     

Telomerase is not an epidermal stem cell marker and is downregulated by calcium

An understanding of the basic mechanisms responsible for the pathogenesis of liver neoplasms is needed in order to develop better therapeutic strategies. The present study utilized a pharmacogenetic mouse model to assess the role of cytochrome P4501A1 (Cyp1a1) in modulating genetic damage to oncogenic and tumor suppressor loci following in utero exposure to the polycyclic aromatic hydrocarbon, 3-methylcholanthrene (MC). Analysis of the Ha-ras, Ki-ras, INK4a and p53 genes was carried out with lysates from paraffin-embedded liver tissue from transplacentally-treated mice. The lysates were subjected to DNA amplification by the PCR technique followed by allele-specific oligonucleotide hybridization screening and SSCP analysis. All of the 26 neoplasms screened (23 hepatocellular carcinomas, two hepatocellular adenomas and one sarcoma) exhibited a GGC-->CGC (GLY13-->ARG13) transversion at the Ki-ras gene locus. None of the tumors had Ki-ras mutations at codon 12 of exon 1. Approximately 12% (3/26) of the liver tumors exhibited point mutations in exon 1 of the INK4a gene, with each of the three tumors exhibiting two point mutations. Analysis of exon 2 of the INK4a gene showed the presence of a CCG-->CTG (PRO73-->LEU73) transition in two of the 26 neoplasms. No mutations were found in exons 1 or 2 of the Ha-ras gene, or in exons 5-8 of the p53 gene. Analysis of tumor RNAs showed overexpression of Ha-ras, cip1 and c-jun in approximately 38% of the liver tumor samples. The results of this study suggest that mutagenic damage to oncogenes and tumor suppressor genes may be critical factors in mediating transplacentally-induced liver tumorigenesis. The fact that Ki-ras mutations were found in all of the tumors suggests that mutation at this gene locus may be an early event in liver tumor pathogenesis, while mutation in tumor suppressor genes may occur later during tumor progression. These combined results are consistent with the pathogenesis of cancer in humans.     

High incidence of the CD8/9 (+G) beta 0-thalassemia mutation in Spain

BACKGROUND AND OBJECTIVE: In Spain, as in other Mediterranean regions the most common beta-thalassemia mutations are due to point mutations in gene regions that are critical for production of mRNA, such as [IVS-I-nt1 (G-->A), IVS-I-nt6 (T-->C), IVS-I-nt110 (G-->A)] which interrupt normal RNA processing or nonsense mutations [CD39 (C-->T)] which interrupt the translation of mRNA. The frameshift mutation CD8/9 (+G) is a very common allele in Asian Indians but is rare in the Mediterranean regions in which isolated alleles with this mutation have been found in Israel, Greece, Portugal and Turkey. DESIGN AND METHODS: We performed a molecular analysis of 175 chromosomes corresponding to 233 beta-thalassemia patients (221 heterozygous, 10 homozygous and 2 compound heterozygous) who belong to 169 Spanish families. The study of beta-thalassemia was made by PCR-ARMS, the alpha genes by Southern blot, the phenotype of Hb Lepore by enzymatic amplification and the presence of -158 gamma G C-->T mutation by PCR and digestion with the restriction enzyme XmnL. RESULTS: Twenty of these 233 patients showed the beta-thalassemia mutation CD8/9 (+G) (17 were heterozygous, 2 homozygous and in one patient the mutation was associated with a structural variant Hb Lepore Boston). INTERPRETATION AND CONCLUSIONS: These data reveal the heterogeneity of beta-thalassemia in Spain and the relatively high frequency (8.6%) of the frameshift mutation CD8/9 (+G). It is surprising that homozygotes for beta zero-thalassemia due to this mutation with very high Hb F values (around 90%) present a phenotype of intermediate thalassemia.     

Analysis of mutational specificity induced by heterocyclic amines in the lacZ gene of Escherichia coli

Mutational spectra induced by different heterocyclic amines were characterized and compared with those obtained from diethylnitrosamine and N-methyl-N-nitrosourea. Mutation classes were identified by means of a series of mutant lacZ genes in F' episomes in Escherichia coli engineered to detect specifically each of two transitions, four transversions and five kinds of frameshift events. More than 99.5% of the mutations induced by heterocyclic amines were frameshift mutations. -2(C.G-G.C) frameshifts were favored over other types, such as +1(G.C), -1(G.C), +1(A.T) and -1(A.T), except when 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) was administered. -1(G.C) and +1(G.C) frameshifts predominate following Trp-P-1 treatment. A small number of G.C-->T.A transversions were induced by the treatment with 2-amino-3,4-dimethylimidazo[4,5-f]quinoline as well as with several other heterocyclic amines examined. Since G.C-->T.A transversions, but not frameshift mutations, are reported to play a role in heterocyclic amine-induced activation of the c-Ha-ras protooncogene or inactivation of the p53 tumor suppressor gene, the low level of base substitutions, particularly G.C-->T.A transversions, may represent a partial explanation for the relatively modest carcinogenic activity of heterocyclic amines, despite their extraordinarily strong mutagenicity in the Salmonella mutation assay.     

A point mutation associated with a severe phenotype of neurofibromatosis 2

Neurofibromatosis 2 (NF2) is an autosomal dominant disease characterized by bilateral vestibular schwannomas and other nonmalignant tumors of the brain, spinal cord, and peripheral nerves. Although the average age of onset of NF2 is 20 years, some individuals may become symptomatic in childhood. We studied 5 unrelated NF2 patients who became symptomatic before age 13. All 5 had multiple tumors in addition to vestibular schwannoma, and none had a positive family history. Sequence analysis of the NF2 gene revealed identical nonsense mutation of exon 6 in 3 patients. Because this mutation destroys a restriction enzyme recognition site, genomic DNA from the 2 other children was directly tested for this change and identical alterations were detected. Although the work of our laboratory and others has not, in general, detected identical mutations in unrelated patients, this mutation seems to occur particularly frequently in the pediatric population and thus may be associated with an especially severe phenotype. Restriction analysis in children with NF2 may be a cost effective way of identifying their mutation. Further work is needed to characterize the effects of this change on the NF2 protein product and its relationship to this severe phenotype.     

A novel insertional mutation in loricrin in Vohwinkel's Keratoderma

A mutation in the gene encoding loricrin has recently been reported in a subset of patients with Vohwinkel's Keratoderma manifesting an associated ichthyosiform dermatosis. We have studied a further kindred with this clinical phenotype. Microsatellite marker analysis was consistent with linkage to chromosome 1q21 and direct sequencing of loricrin identified a heterozygous mutation with an insertion of a T residue at codon 209. This mutation is predicted to produce a mutant protein with a frameshift of its terminal 107 amino acids and to be 22 amino acids longer than the wild-type protein due to a delayed termination codon. The only previously reported mutation is a G insertion producing a frameshift after codon 231. The novel mutation we report is likely to have a similar functional effect on cornified envelope formation, with disturbance of transglutaminase-mediated cross-linking of envelope components, and serves to confirm the predicted role of insertional mutations in Vohwinkel's Keratoderma associated with ichthyosis.     

Molecular basis of hereditary fructose intolerance in Italy: identification of two novel mutations in the aldolase B gene

We screened the aldolase B gene in 14 unrelated Italian patients with hereditary fructose intolerance (HFI), and found two novel disease related mutations: a single nucleotide deletion in exon 2 (delta A20) that leads to an early stop codon, and a C-->T transition in exon 8 that substitutes an Arg with a Trp residue at codon 303 (R303W).