Two new Gaucher disease mutations

Recently, a mutation at nucleotide 1193 of the glucocerebrosidase gene was described in a patient with type 1 Gaucher disease. This mutation destroys a TaqI site in a polymerase chain reaction (PCR)-amplified fragment. We used digestion with this enzyme to screen DNA samples from Gaucher disease patients representing 23 previously unidentified alleles and discovered that this site had been destroyed in three samples. However, the mutation that caused this change proved to be a CT substitution at cDNA nucleotide 1192 (Genomic 5408; ³⁵⁹ArgEnd). Fortuitously, another TaqI site was destroyed by a different mutation, a GA mutation at nt 1312 (Genomic 5927; ³⁹⁹AspAsn). Both of these mutations were functionally severe in that they were associated with type 2 (acute neuronopathic) Gaucher disease.     

The Ca2+-sensing receptor gene (PCAR1) mutation T151M in isolated autosomal dominant hypoparathyroidism

Isolated autosomal dominant hypoparathyroidism is a heterogeneous disorder characterized by parathyroid hormone (PTH) deficiency, hypocalcemia and hyperphosphatemia. The candidate gene approach was used to study a large Norwegian family. The loci for the PTH gene, PTH receptor gene and RET protooncogene were excluded using dinucleotide markers and restriction fragment length polymorphism analysis. Complete cosegregation of this trait was found with the chromosomal region 3q13, using the short tandem repeat markers D3S1267, D3S1269, D3S1303, D3S1518, and RHO. This region contains the candidate locus for the Ca²⁺-sensing receptor (PCAR1). By single-strand conformation polymorphism (SSCP) analysis of all PCAR1 exons followed by automated sequencing, we identified a C to T transition in exon 2 (cDNA position 452) on the mutant allele in the family. The mutation predicts a substitution of Thr to Met in amino acid position 151 (T151M). A StyI restriction site created by the nucleotide substitution was used to confirm the mutation on all alleles, as well as to exclude it among 100 normal alleles (blood donors). SSCP analysis also identified a novel polymorphism of PCAR1 intron 4 (1609–88t→c) on normal alleles.The T151M mutation is located in the extracellular N-terminal domain of PCAR1, which belongs to the superfamily of G protein-coupled receptors. We suggest that this is a gain-of-function mutation that increases the sensitivity of the receptor to [Ca²⁺], thereby decreasing the calcium set point. Received: 29 September 1995 / Revised: 19 January 1996     

Sex-limited genetic variation in a mouse salivary protein

This report describes a gene which influences the electrophoretic mobility of a protein in the salivas of adult mice. Three categories of phenotype have been observed: the two single-banded types, F (Fast) and S (Slow), and the two-banded type, SF (Slow-Fast), with the two bands represented in varying proportions. All females, regardless of age or strain, and all males before puberty show only the F phenotype. Males of the BALB/c and C57BL/6J strains show the F phenotype throughout puberty and adult life, whereas males of the C3H/St and C57BL/KsJ strains show the SF phenotype in puberty and the S phenotype in adult life. We have designated this variation the sex-limited saliva pattern (Ssp). The results from genetic crosses indicate that the variation among the strains is determined by an autosomal locus, Ssp, with two alleles, Ssp ^S andSsp ^F ,where Ssp ^S is dominant to Ssp ^F .Testosterone treatment can accelerate the acquisition of the S type in males of the strains C3H/St and C57BL/KsJ and also induces that phenotype in C3H/St females and C57BL/6J males. Thus it appears that the observed strain-specific differences reflect a genetic variation in androgen levels and/or androgen sensitivity rather than variation in a structural gene.This study was supported in part by PHS Research Grant 5 RO1 AM21177 and by the Indiana University Human Genetics Center (PHS PO1 GM21054). The preliminary work was done during a 1-month visit by RCK to the Institute of Ecology and Genetics which was supported by the University of Aarhus. This is publication No. 80-18 from the Department of Medical Genetics. RCK was supported by PHS Career Development Award 1 KO4 AM00284. SRD was supported by PHS General Medical Training Grant T32 GM07468.     

Molecular analysis of the gene for the human vitamin-D-binding protein (group-specific component): allelic differences of the common genetic GC types

Summary DNA sequence analysis of the polymerase chain reaction products, including the coding region for amino acids 416 and 420, of the vitamin-D-binding protein (DBP, group-specific component, GC) shows allelespecific differences. The GC2 and GC1F phenotypes have an aspartic acid residue at amino acid position 416, whereas the GC1S phenotype has a glutamic acid at this position. In the GC2 phenotype, amino acid 420 is a lysine residue, and in the both common GC1 phenotypes, it is a threonine residue. The nucleotide exchanges involve a HaeIII (position 416) and a StyI (position 420) restriction site: the HaeIII restriction site is specific for the GC^*1S allele and the StyI restriction site is specific for the GC^*2 allele. We have tested 140 individual genomic DNA samples for the HaeIII site and 148 samples for the StyI site by restriction fragment length polymorphism (RFLP) analysis with a DBP-specific direct genomic DNA probe, and have compared these findings with the GC phenotype classification, by isoelectric focusing (IEF) of the corresponding plasma. The results of the HaeIII RFLP analysis and the IEF typing were in complete agreement. By using our DNA probe, we could disclose, in addition to the StyI site at amino acid position 420, two further StyI site downstream: one was specific for the GC^*1S allele and another for the GC^*1F allele. In 147 samples, there was agreement between the IEF GC typing and the analysis of the StyI restriction sites. In a single case, the observed result of the StyI-digest differed from the result expected after IEF classification: homozygous GC 1F-1F by IEF and heterozygous by StyI RFLP analysis. We discuss this finding as a recombination event or a possible silent allele in IEF typing. The GC polymorphism revealed by Southern blot analysis of StyI-digests provides an informative DNA marker system for chromosome 4q11–q13.     

Multiple gene action determining a mouse salivary protein phenotype: Identification of the structural gene for androgen binding protein (Abp)

A novel variation in electrophoretic phenotype is described for a mouse salivary androgen binding protein (Abp). Crosses show that the variation is inherited in an autosomal codominant manner and protein characterization studies show that the variant Abp differs in isoelectric point from the common form of the protein. Those observations suggest that the variation involves the structural gene for the mouse salivary Abp. The genetic studies also show that the electrophoretic mobility of the variant Abp can be influenced by the sex-limited saliva pattern (Ssp) gene. The Ssp ^S allele alters the electrophoretic mobility of Abp in males at puberty or in females which have received exogenous testosterone [Karn, R. C., Dlouhy, S. R., Springer, K. R., Hjorth, J. P., and Nielsen, J. T. (1982). Biochem Genet. 20:493]. This study shows that Abp and Ssp are distinct genes which are not closely linked and that Ssp ^S is trans active in F₁ (Abp ^a /Abp ^b , Ssp ^S /Ssp ^F ) males.SRD was supported in part by PHS General Medical Training Grant T32 GMO7468 and the Indiana University School of Medicine Research Program in Academic Medicine. RCK was supported in part by PHS Career Development Award 1 KO4 AMOO284.     

The polymorphism of the plasma inter-α-trypsin inhibitor (ITI) and its relationship to the heavy chain H1 subunit gene (ITIH1) at 3p211-212

We investigated the ITI protein polymorphism in linkage analysis, usingDraI andSstI as restriction fragment length polymorphism (RFLP) markers for the ITIH1 gene. Isoelectric focusing (IEF) classification from 76 individual plasma samples and RFLP analysis from the corresponding DNA preparations disclosed linkage disequilibrium between the phenotypic IEF patterns of the two common ITI alleles, ITI^*1 and ITI^*2, and the diallelic DNA polymorphisms of two ITIH1 RFLPs, represented byDraI 4.0 kb andDraI 2.4 + 1.6 kb, and bySstI 6.7 kb andSstI 6.0 + 0.7 kb, for the ITI 1 and ITI 2 IEF phenotypes, respectively, and byDraI 4.0/2.4 + 1.6 kb andSstI 6.7/6.0 + 0.7 kb for the heterozygous ITI 1–2 IEF phenotype. Linked segregation between either of the RFLPs and the polymorphic ITI plasma protein locus has been established in nine informative family pedigrees. The less frequent allele in Europeans, ITI^*3, is not represented by a further allelic restriction fragment in either RFLP. The significant linkage disequilibrium observed in this genetic study indicates that the ITI locus, with the alleles ITI^*1 and ITI^*2, must be close to, or reside within, the ITIH1 gene. The diallelic ITI protein polymorphism therefore provides an informative phenotypic marker system for chromosome 3p211-212.     

Atypical segregation of esterase D: Evidence of a rare “silent” allele EsD 0

Summary Electrophoretic study of esterase D in 1027 mother-child pairs showed an atypical segregation of EsD alleles in one pair. The family analysis confirmed the evidence of a silent gene (EsD ⁰), which was observed in child, mother and grandfather. R banding of the metaphasal chromosomes revealed the normal appearance of the No. 13 pair, and no deletion of homologues No. 13 was observed in this family.     

Isoelectric focusing studies of human red cell esterase D: Evidence for polymorphic occurrence of a new allele EsD 7 in Japanese

Summary The isoelectric focusing study of esterase D in Japanese revealed evidence of a new polymorphic allele (EsD ⁷) which is difficult to find by conventional starch gel electrophoresis only. A comparison with the occurrence of a subdivision of EsD ² in Caucasians (EsD ⁵) suggests a remarkable difference in allele distribution of esterase D among races. Quantitative analysis showed a relatively low value of enzyme activity for this new allele. It is therefore emphasized that in addition to conventional electrophoresis, enzyme assay and further detection by isoelectric focusing are essential in analyzing the esterase D system.     

Bayesian approach to searching for susceptibility genes: Gc2 and EsD1 alleles and multiple sclerosis

Multiple sclerosis (MS) is one of the most common causes of neurological disability in early adulthood. The current literature is interested in identifying biological or DNA markers associated with genetic susceptibility to MS. The aim of this study is to investigate, by means of Bayesian statistical inference, whether the presence of Gc2 (Gc = group-specific component) and/or EsD1 (EsD = esterase D) alleles affects MS susceptibility. Gc and EsD are two classical genetic markers, being the first a serum protein polymorphism, the latter an isoenzyme polymorphism. The interest of the proposed statistical approach of searching for MS susceptibility genes relies on the analysis of two different functions, one function being inferred from our results on 56 unrelated patients from central Italy affected by MS, the other one from Italian and worldwide epidemiological data. The graphical analysis suggests that MS susceptibility is influenced by both Gc2 and EsD1 alleles; and EsD1 allele is more informative than Gc2. These results point out the advantages of the Bayesian approach in searching for susceptibility genes. Furthermore, the significant association between the considered alleles and the susceptibility to MS suggests possible hypotheses about the pathogenesis of the disease.     

Characterization of Mutations at the Mouse Phenylalanine Hydroxylase Locus

Two genetic mouse models for human phenylketonuria have been characterized by DNA sequence analysis. For each, a distinct mutation was identified within the protein coding sequence of the phenylalanine hydroxylase gene. This establishes that the mutated locus is the same as that causing human phenylketonuria and allows a comparison between these mouse phenylketonuria models and the human disease. A genotype/phenotype relationship that is strikingly similar to the human disease emerges, underscoring the similarity of phenylketonuria in mouse and man. InPAH^ENU1,the phenotype is mild. ThePah^enu1mutation predicts a conservative valine to alanine amino acid substitution and is located in exon 3, a gene region where serious mutations are rare in humans. InPAH^ENU2,the phenotype is severe. ThePah^enu2mutation predicts a radical phenylalanine to serine substitution and is located in exon 7, a gene region where serious mutations are common in humans. InPAH^ENU2,the sequence information was used to devise a direct genotyping system based on the creation of a newAlw26I restriction endonuclease site.     

Tyrosinemia type 1 — complex splicing defects and a missense mutation in the fumarylacetoacetase gene

Two mutations are reported in six tyrosinemia type 1 patients from northern Europe. In four patients, a G to A transition at nucleotide position 1009 (G¹⁰⁰⁹A) of the fumarylacetoacetase (FAH) coding sequence caused aberrant splicing by introducing an acceptor splice site within exon 12, thereby deleting the first 50 nucleotides of this exon. The following exon-intron boundary was frequently missed, and a cryptic donor splice site within intron 12 caused a partial intron 12 retention of 105 bp. This point mutation alternatively gave a glycine 337 to serine substitution in instances of correct splicing. The mutation is rapidly detected by PvuII digestion of polymerase chain reaction (PCR)-amplified genomic DNA. Another mutation, g⁺⁵a in the intron 12 donor splice site consensus sequence (IVS12 g⁺⁵a), was found in five of the patients. This caused alternative splicing with retention of the first 105 nucleotides of intron 12, exon 12 skipping, and a combined deletion of exons 12 and 13. Rapid detection of this mutation is achieved by restriction digestion of PCR-amplified genomic DNA; a mismatch primer combined with the point mutation creates a Tru9I restriction site. One patient who was homozygous for the G¹⁰⁰⁹A mutation had a chronic form of tyrosinemia. Three patients were combined heterozygotes for G¹⁰⁰⁹A and TVS12 g⁺⁵a. Their clinical phenotypes varied from acute to chronic, indicating the impact of background genes and/or external factors on the presentation of typrosinemia type 1.     

PCR amplification following restriction to detect site-specific DNA methylation

A procedure to test for DNA methylation at sites recognized by methylation-sensitive restriction endonucleases is described. The procedure is based on the assumption that the polymerase chain reaction (PCR) will amplify sequences between two primers only if the target DNA is intact after digestion. A carrot (Daucus carota) cell line that is heterozygous for two sequenced alleles ofDc8, a gene which is expressed during the later stages of embryogenesis provided an ideal source of DNA for developing and testing protocols. The promoters of the two alleles differs significantly in length between two sites used for primers, and only one promoter has a GATC (Sau 3A1 orMbo I) site. This allowed development of a protocol where only the sequence lacking the GATC site was amplified to detectable levels following digestion of DNA withMbo I which is insensitive to symmetric methylation with^m4C or^m5C.     

G-6-PD polymorphism and racial admixture in the Cuban population

Summary Esterase D phenotypes were determined in 1082 non-related individuals from the western region of Germany by agarose-gel electrophoresis. Gene frequencies were compared with previous data and all European populations studied so far agreed with the Hardy-Weinberg equilibrium. Mean gene frequencies for Europeans are: EsD ¹ 0.8888, EsD ² 0.1112.     

Molecular diagnosis of the transthyretin (TTR) Met111 mutation in familial amyloid cardiomyopathy of Danish origin

Summary Familial amyloid cardiomyopathy in a Danish kindred is associated with a specific mutation (Met for Leu¹¹¹) in the transthyretin (TTR) gene, causing the loss of a recognition site for the restriction enzyme DdeI in the gene. We describe a diagnostic test for the molecular detection of this mutation. A sequence of the TTR gene containing the mutation was amplified by the polymerase chain reaction from isolated genomic DNA of two affected patients and several controls. DdeI digestion of the amplified DNA from the patients revealed 3 bands by gel-electrophoresis, whereas amplified DNA of the controls showed only 2 bands, consistent with complete digestion. Thus, the assumed heterozygous TTR Met¹¹¹ mutation was confirmed in the affected patients.     

Somatic cell variants of H-2k: b: A point mutation in the first extracellular domain results in altered immune recognition

A cell-surface-associated variant H-2K product was expressed by an Abelson virus-induced pre-B-cell line after chemical mutagenesis with ethyl methane sulfonate. The variant cell line (R8.313) was previously demonstrated to have altered allodeterminants in K^b as demonstrated by both K^b-specific monoclonal antibody binding and alloreactive cytotoxic T lymphocyte (CTL) cytolysis. The mutant H-2K ^b gene from R8.313 was cloned and characterized in detail. DNA sequence analysis of the region of the gene corresponding to the three extracellular domains identified a single point mutation resulting in a leucine-to-phenylalanine substitution at amino acid residue 82. The site of mutation within the 1 domain was confirmed by oligonucleotide hybridization analysis. Mouse L-cell fibroblasts transfected with the mutant gene were recognized with the same monoclonal antibody binding and CTL lytic pattern as the R8.313 cell line, confirming that the altered phenotype of the mutant cell line was due to a point mutation in the H-2K ^b gene. These data further extend the hypothesis that the region of amino acid residues 70–90 in the 1 domain is important in the formation of both antibody and CTL-defined recognition structures on major histocompatibility complex class I molecules.     

Chocolate coated cats: TYRP1 mutations for brown color in domestic cats

Brown coat color phenotypes caused by mutations in tyrosinase-related protein-1 (TYRP1) are recognized in many mammals. Brown variations are also recognized in the domestic cat, but the causative mutations are unknown. In cats, Brown, B, has a suggested allelic series, B > b > b^l. The B allele is normal wild-type black coloration. Cats with the brown variation genotypes, bb or bb^l, are supposedly phenotypically chocolate (aka chestnut) and the light brown genotype, b^lb^l, are supposedly phenotypically cinnamon (aka red). The complete coding sequence of feline TYRP1 and a portion of the 5′ UTR was analyzed by direct sequencing of genomic DNA of wild-type and brown color variant cats. Sixteen single nucleotide polymorphisms (SNPs) were identified. Eight SNPs were in the coding regions, six are silent mutations. Two exon 2 on mutations cause amino acid changes. The C to T nonsense mutation at position 298 causes an arginine at amino acid 100 to be replaced by the opal (UGA) stop codon. This mutation is consistent with the cinnamon phenotype and is the putative light brown, b^l, mutation. An intron 6 mutation that potentially disrupts the exon 6 downstream splice-donor recognition site is associated with the chocolate phenotype and is the putative brown, b, mutation. The allelic series was confirmed by segregation and sequence analyses. Three microsatellite makers had significant linkage to the brown phenotype and two for the TYRP1 mutations in a 60-member pedigree. These mutations could be used to identify carriers of brown phenotypes in the domestic cat.     

Sequencing and Amplified Restriction Fragment Length Polymorphism Analysis of Ribonucleotide Reductase Large Subunit Gene of the White Spot Syndrome Virus in Blue Crab (Callinectes sapidus) from American Coastal Waters

In the present study, the existence of white spot syndrome virus (WSSV) in blue crab (Callinectes sapidus) collected from 3 different American coastal waters (New York, New Jersey, and Texas) was confirmed by 2-step diagnostic polymerase chain reaction and in situ hybridization analysis. When geographic isolates were also compared using a gene that encodes the WSSV ribonucleotide reductase large subunit RR1 (WSSV rr1), a C¹⁶⁶¹-to-T point mutation was found in the New Jersey WSSV isolated. This point mutation, which resulted in the creation of an additional RsaI endonuclease recognition site, was not found in the WSSV from the New York and Texas blue crab samples, or in the WSSV Taiwan isolate, or in any of the other WSSV geographical isolates for which data are available. WSSV rr1-specific RsaI amplified restriction fragment length polymorphism of an amplified 1156-bp fragment thus distinguished the New Jersey blue crab samples from the other WSSV isolates. Received June 29, 2000; accepted October 11, 2000     

Molecular basis of the apolipoprotein H (β2-glycoprotein I) protein polymorphism

Apolipoprotein H (apoH, protein; APOH, gene) is considered to be an essential cofactor for the binding of certain antiphospholipid autoantibodies to anionic phospholipids. APOH exhibits a genetically determined structural polymorphism due to the presence of three common alleles (APOH*1, APOH*2 and APOH*3 ) detectable by isoelectric focusing (IEF) and immunoblotting. The APOH*3 allele can be further characterized into two subtypes, APOH*3^W and APOH*3^B, based upon its reactivity with monoclonal antibody 3D11. In this study we have determined the molecular basis of the APOH protein polymorphism and its distribution in three large U.S. population samples comprising 661 non-Hispanic whites, 444 Hispanics and 422 blacks. By direct DNA sequencing of PCR amplified fragments corresponding to the eight APOH exons, we identified two missense mutations that correspond to the APOH*1 and APOH*3^W alleles. A missense mutation (G→A) in exon 3, which alters amino acid Ser to Asn at codon 88 and creates a restriction site for TSP509 I, was present in all APOH*1 allele carriers. A second missense mutation (G→C) at codon 316 in exon 8, which replaces amino acid Trp with Ser and creates a restriction site for BSTBI, was present in all APOH*3^W carriers. The distribution of the Ser 88 Asn and Trp 316 Ser mutations was significantly different between the three racial groups. The frequency of the Asn-88 allele was 0.011, 0.043, and 0.056 in blacks, Hispanics and non-Hispanic whites, respectively. While the Ser-316 allele was observed sporadically in blacks (0.008), it was present at a polymorphic frequency in Hispanics (0.027) and non-Hispanic whites (0.059). The identification of the molecular basis of the APOH protein polymorphism will help to elucidate the structural – functional relationship of apoH in the production of antiphospholipid autoantibodies. Received: 20 November 1996 / Accepted: 13 February 1997     

Nature and frequency of mutations in the argininosuccinate synthetase gene that cause classical citrullinemia

Citrullinemia is an autosomal recessive disorder caused by a genetic deficiency of argininosuccinate synthetase (ASS). So far 20 mutations in ASS mRNA have been identified in human classical citrullinemia, including 14 single base changes causing missense mutations in the coding sequence of the enzyme, 4 mutations associated with an absence of exons 5, 6, 7, or 13 in mRNA, 1 mutation with a deletion of the first 7 bases in exon 16 (which is caused by abnormal splicing), and 1 mutation with an insertion of 37 bases between the exon 15 and 16 regions in mRNA. In order to identify the abnormality in the ASS gene causing the exon 7 and 13 deletion mutations and the 37-base insertion mutation between exons 15 and 16 in mRNA, and to establish a DNA diagnostic test, we isolated and sequenced the genomic DNA surrounding each exon. The absence of exon 7 or 13 in ASS mRNA resulted from abnormal splicing caused by a single base change in the intron region: IVS-6^–2 (a transition of A to G at the second nucleotide position within the 3 splice cleavage site of intron 6) and IVS-13⁺⁵ (a transition of G to A at the fifth nucleotide position within the 5 splice cleavage site of intron 13), respectively. The IVS-6^–2 mutation resulted in the creation of an MspI restriction site. DNA diagnostic analysis of 33 Japanese alleles with classical citrullinemia showed that 19 alleles had the IVS-6^–2 mutation (over 50% of the mutated alleles in Japanese patients). It was thus confirmed that one mutation is predominant in Japan. This differs from the situation in the USA where there is far greater heterogeneity. The insertion mutation in mRNA on the other hand resulted from abnormal splicing caused by a 13-bp deletion at the splice-junction between exon 15 and intron 15. The deletion had a short direct repeat (CTCAGG) at the breakpoint junction and presumably resulted from slipped mispairing.     

Human red cell esterase D polymorphism in Denmark, its use in paternity cases and the description of a new phenotype

Red cell esterase D (EsD) phenotypes were determined in a Danish population sample of 3,116 unrelated adults by starch-gel electrophoresis. A new phenotype was discovered, which appeared to be determined by the EsD1 allele and a new allele EsDCph. The gene frequencies observed were EsD1 = 0.9007, EsD2 = 0.0992, EsDCph = 0.0001. Investigation of 1,111 mother-child pairs and 59 families with 157 offspring added further support to the genetic model of two common alleles at an autosomal locus. The applicability of the EsD polymorphism to paternity testing was investigated on 960 cases of disputed paternity. An estimate of the EsD null allele frequency (0.001) in European populations was made on the basis of observations made on 5,864 mother/child combinations and 762 matings with 1,882 offspring. The influence of this allele on the reliability of exclusions of paternity was determined.