A pseudodeficiency allele common in non-Jewish Tay-Sachs carriers: Implications for carrier screening

Deficiency of beta-hexosaminidase A (Hex A) activity typically results in Tay-Sachs disease. However, healthy subjects found to be deficient in Hex A activity (i.e., pseudodeficient) by means of in vitro biochemical tests have been described. We analyzed the HEXA gene of one pseudodeficient subject and identified both a C739-to-T substitution that changes Arg247----Trp on one allele and a previously identified Tay-Sachs disease mutation on the second allele. Six additional pseudodeficient subjects were found to have the C739-to-T mutation. This allele accounted for 32% (20/62) of non-Jewish enzyme-defined Tay-Sachs disease carriers but for none of 36 Jewish enzyme-defined carriers who did not have one of three known mutations common to this group. The C739-to-T allele, together with a "true" Tay-Sachs disease allele, causes Hex A pseudodeficiency. Given both the large proportion of non-Jewish carriers with this allele and that standard biochemical screening cannot differentiate between heterozygotes for the C739-to-T mutations and Tay-Sachs disease carriers, DNA testing for this mutation in at-risk couples is essential. This could prevent unnecessary or incorrect prenatal diagnoses.     

A second mutation associated with apparent beta-hexosaminidase A pseudodeficiency: identification and frequency estimation.

Deficient activity of beta-hexosaminidase A (Hex A), resulting from mutations in the HEXA gene, typically causes Tay-Sachs disease. However, healthy individuals lacking Hex A activity against synthetic substrates (i.e., individuals who are pseudodeficient) have been described. Recently, an apparently benign C739-to-T (Arg247Trp) mutation was found among individuals with Hex A levels indistinguishable from those of carriers of Tay-Sachs disease. This allele, when in compound heterozygosity with a second "disease-causing" allele, results in Hex A pseudodeficiency. We examined the HEXA gene of a healthy 42-year-old who was Hex A deficient but did not have the C739-to-T mutation. The HEXA exons were PCR amplified, and the products were analyzed for mutations by using restriction-enzyme digestion or single-strand gel electrophoresis. A G805-to-A (Gly269Ser) mutation associated with adult-onset GM2 gangliosidosis was found on one chromosome. A new mutation, C745-to-T (Arg249Trp), was identified on the second chromosome. This mutation was detected in an additional 4/63 (6%) non-Jewish and 0/218 Ashkenazi Jewish enzyme-defined carriers. Although the Arg249Trp change may result in a late-onset form of GM2 gangliosidosis, any phenotype must be very mild. This new mutation and the benign C739-to-T mutation together account for approximately 38% of non-Jewish enzyme-defined carriers. Because carriers of the C739-to-T and C745-to-T mutations cannot be differentiated from carriers of disease-causing alleles by using the classical biochemical screening approaches, DNA-based analyses for these mutations should be offered for non-Jewish enzyme-defined heterozygotes, before definitive counseling is provided.     

Glucocerebrosidase mutations in Gaucher disease.

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.     

The hemochromatosis 845 G-->A and 187 C-->G mutations: prevalence in non-Caucasian populations.

Hemochromatosis, the inherited disorder of iron metabolism, leads, if untreated, to progressive iron overload and premature death. The hemochromatosis gene, HFE, recently has been identified, and characterization of this gene has shown that it contains two mutations that result in amino acid substitutions-cDNA nucleotides 845 G-->A (C282Y) and 187 C-->G (H63D). Although hemochromatosis is common in Caucasians, affecting >=1/300 individuals of northern European origin, it has not been recognized in other populations. The present study used PCR and restriction-enzyme digestion to analyze the frequency of the 845 G-->A and 187 C-->G mutations in HLA-typed samples from non-Caucasian populations, comprising Australian Aboriginal, Chinese, and Pacific Islanders. Results showed that the 845 G-->A mutation was present in these populations (allele frequency 0.32%), and, furthermore, it was always seen in conjunction with HLA haplotypes common in Caucasians, suggesting that 845 G-->A may have been introduced into these populations by Caucasian admixture. 187 C-->G was present at an allele frequency of 2.68% in the two populations analyzed (Australian Aboriginal and Chinese). In the Australian Aboriginal samples, 187 C-->G was found to be associated with HLA haplotypes common in Caucasians, suggesting that it was introduced by recent admixture. In the Chinese samples analyzed, 187 C-->G was present in association with a wide variety of HLA haplotypes, showing this mutation to be widespread and likely to predate the more genetically restricted 845 G-->A mutation.     

Mutational analyses of Tay-Sachs disease: studies on Tay-Sachs carriers of French Canadian background living in New England.

Tay-Sachs disease (TSD) results from mutations in HEXA that cause Hex A deficiency. Heterozygote-screening programs have been applied in groups with an increased TSD incidence, such as Ashkenazi Jews and French Canadians in Quebec. These programs are complicated by benign mutations that cause apparent Hex A deficiency but not TSD. Benign mutations account for only approximately 2% of Jewish and approximately 36% of non-Jewish enzyme-defined carriers. A carrier frequency of 1/53 (n = 1,434) was found in an ongoing prospective analysis of persons of French Canadian background living in New England by using an enzyme-based assay. DNA from enzyme-defined carriers from this population was analyzed to determine the molecular basis of Hex A deficiency. Samples (36) were tested for common mutations, and samples that were negative for these were screened for uncommon or novel mutations by using SSCP analysis. Exons showing mobility shifts were sequenced, and most mutations were confirmed by restriction enzyme digestion. Known disease-causing mutations were found in nine samples (four had a 7.6-kb deletion found in 80% of French Canadian TSD alleles), and known benign mutations were found in four samples. Seven novel changes were identified, including G748A in four samples. The molecular basis of Hex A deficiency in this carrier population differs from that of French Canadian TSD patients. Screening centers should be aware of the presence of benign mutations among U.S. French Canadians or Franco-Americans.(ABSTRACT TRUNCATED AT 250 WORDS)     

A pseudodeficiency allele (D152N) of the human beta-glucuronidase gene.

We present evidence that a 480G-->A transition in the coding region of the beta-glucuronidase gene, which results in an aspartic-acid-to-asparagine substitution at amino acid position 152 (D152N), produces a pseudodeficiency allele (GUSBp) that leads to greatly reduced levels of beta-glucuronidase activity without apparent deleterious consequences. The 480G-->A mutation was found initially in the pseudodeficient mother of a child with mucopolysaccharidosis VII (MPSVII), but it was not on her disease-causing allele, which carried the L176F mutation. The 480G-->A change was also present in an unrelated individual with another MPSVII allele who had unusually low beta-glucuronidase activity, but whose clinical symptoms were probably unrelated to beta-glucuronidase deficiency. This individual also had an R357X mutation, probably on his second allele. We screened 100 unrelated normal individuals for the 480G-->A mutation with a PCR method and detected one carrier. Reduced beta-glucuronidase activity following transfection of COS cells with the D152N cDNA supported the causal relationship between the D152N allele and pseudodeficiency. The mutation reduced the fraction of expressed enzyme that was secreted. Pulse-chase experiments indicated that the reduced activity in COS cells was due to accelerated intracellular turnover of the D152N enzyme. They also suggested that a potential glycosylation site created by the mutation is utilized in approximately 50% of the enzyme expressed.     

Canavan disease: mutations among Jewish and non-jewish patients.

Canavan disease is an autosomal recessive leukodystrophy caused by the deficiency of aspartoacylase (ASPA). Sixty-four probands were analyzed for mutations in the ASPA gene. Three point mutations--693C-->A, 854A-->C, and 914C-->A--were identified in the coding sequence. The 693C-->A and 914C-->A base changes, resulting in nonsense tyr231-->ter and missense ala305-->glu mutations, respectively, lead to complete loss of ASPA activity in in vitro expression studies. The 854A-->C transversion converted glu to ala in codon 285. The glu285-->ala mutant ASPA has 2.5% of the activity expressed by the wild-type enzyme. A fourth mutation, 433 --2(A-->G) transition, was identified at the splice-acceptor site in intron 2. The splice-site mutation would lead to skipping of exon 3, accompanied by a frameshift, and thus would produce aberrant ASPA. Of the 128 unrelated Canavan chromosomes analyzed, 88 were from probands of Ashkenazi Jewish descent. The glu285-->ala mutation was predominant (82.9%) in this population, followed by the tyr231-->ter (14.8%) and 433 --2(A-->G) (1.1%) mutations. The three mutations account for 98.8% of the Canavan chromosomes of Ashkenazi Jewish origin. The ala305-->glu mutation was found exclusively in non-Jewish probands of European descent and constituted 60% of the 40 mutant chromosomes. Predominant occurrence of certain mutations among Ashkenazi Jewish and non-Jewish patients with Canavan disease would suggest a founding-father effect in propagation of these mutant chromosomes.     

Mutation and polymorphism analysis of the human homogentisate 1, 2-dioxygenase gene in alkaptonuria patients.

Alkaptonuria (AKU), a rare hereditary disorder of phenylalanine and tyrosine catabolism, was the first disease to be interpreted as an inborn error of metabolism. AKU patients are deficient for homogentisate 1,2 dioxygenase (HGO); this deficiency causes homogentisic aciduria, ochronosis, and arthritis. We cloned the human HGO gene and characterized two loss-of-function mutations, P230S and V300G, in the HGO gene in AKU patients. Here we report haplotype and mutational analysis of the HGO gene in 29 novel AKU chromosomes. We identified 12 novel mutations: 8 (E42A, W97G, D153G, S189I, I216T, R225H, F227S, and M368V) missense mutations that result in amino acid substitutions at positions conserved in HGO in different species, 1 (F10fs) frameshift mutation, 2 intronic mutations (IVS9-56G-->A, IVS9-17G-->A), and 1 splice-site mutation (IVS5+1G-->T). We also report characterization of five polymorphic sites in HGO and describe the haplotypic associations of alleles at these sites in normal and AKU chromosomes. One of these sites, HGO-3, is a variable dinucleotide repeat; IVS2+35T/A, IVS5+25T/C, and IVS6+46C/A are intronic sites at which single nucleotide substitutions (dimorphisms) have been detected; and c407T/A is a relatively frequent nucleotide substitution in the coding sequence, exon 4, resulting in an amino acid change (H80Q). These data provide insight into the origin and evolution of the various AKU alleles.     

Cooperative assembly of proteins in the ribosomal GTPase centre demonstrated by their interactions with mutant 23S rRNAs.

The ribosomal protein L11 binds to the region of 23S rRNA associated with the GTPase-dependent steps of protein synthesis. Nucleotides 1054-1107 within this region of the Escherichia coli 23S rRNA gene were mutagenized with bisulphite. Twenty point mutations (G-->A and C-->T transitions) and numerous multiple mutations were generated. Expression of mutant 23S rRNAs in vivo shows that all the mutations detectably alter the phenotype, with effects ranging from a slight growth rate reduction to lack of viability. Temperature sensitivity is conferred by 1071G-->A and 1092C-->U substitutions. These effects are relieved by point mutations at other sites, indicating functional interconnections within the higher order structure of this 23S rRNA region. Several mutations prevent direct binding of r-protein L11 to 23S rRNA in vitro. These mutations are mainly in a short irregular stem (1087-1102) and within a hairpin loop (1068-1072), where the protein probably makes nucleotide contacts. Some of these mutations also interfere with binding of the r-protein complex L10.(L12)4 to an adjacent site on the rRNA. When added together to rRNA, proteins L10.(L12)4 and L11 bind cooperatively to overcome the effects of mutations at 1091 and 1099. The proteins also stimulate each others binding to rRNA mutated at 1087 or 1092, although in these cases binding remains clearly substoichiometric. Surprisingly, none of the mutations prevents incorporation of L11 into ribosomes in vivo, indicating that other, as yet unidentified, factors are involved in the cooperative assembly process.     

Identification of genetic mutations in Japanese patients with fructose-1,6-bisphosphatase deficiency.

Fructose-1,6-bisphosphatase (FBPase) deficiency is an autosomal recessive inherited disorder and may cause sudden unexpected infant death. We reported the first case of molecular diagnosis of FBPase deficiency, using cultured monocytes as a source for FBPase mRNA. In the present study, we confirmed the presence of the same genetic mutation in this patient by amplifying genomic DNA. Molecular analysis was also performed to diagnose another 12 Japanese patients with FBPase deficiency. Four mutations responsible for FBPase deficiency were identified in 10 patients from 8 unrelated families among a total of 13 patients from 11 unrelated families; no mutation was found in the remaining 3 patients from 3 unrelated families. The identified mutations included the mutation reported earlier, with an insertion of one G residue at base 961 in exon 7 (960/961insG) (10 alleles, including 2 alleles in the Japanese family from our previous report [46% of the 22 mutant alleles]), and three novel mutations--a G-->A transition at base 490 in exon 4 (G164S) (3 alleles [14%]), a C-->A transversion at base 530 in exon 4 (A177D) (1 allele [4%]), and a G-->T transversion at base 88 in exon 1 (E30X) (2 alleles [9%]). FBPase proteins with G164S or A177D mutations were enzymatically inactive when purified from E. coli. Another new mutation, a T-->C transition at base 974 in exon 7 (V325A), was found in the same allele with the G164S mutation in one family (one allele) but was not responsible for FBPase deficiency. Our results indicate that the insertion of one G residue at base 961 was associated with a preferential disease-causing alternation in 13 Japanese patients. Our results also indicate accurate carrier detection in eight families (73%) of 11 Japanese patients with FBPase deficiency, in whom mutations in both alleles were identified.     

Autosomal recessive phosphorylase kinase deficiency in liver, caused by mutations in the gene encoding the beta subunit (PHKB).

The association of autosomal recessive phosphorylase kinase deficiency in liver of a 3 1/2-year-old female child with mutations in the gene encoding the common part of the beta subunit of phosphorylase kinase is reported. The proband had a severe deficiency of phosphorylase kinase in liver, while the phosphorylase kinase activity in erythrocytes was only slightly diminished. She had no symptoms of muscle involvement. The complete coding sequences of the liver gamma subunit and of the beta subunit of phosphorylase kinase of the proband were analyzed for the presence of mutations, by either reverse-transcribed PCR or SSCP analysis. Three deviations from the normal sequence were found in the region encoding the common part of the beta subunit of phosphorylase kinase-namely, a 1827G-->A (W609X) transition, a 2309A-->G (Y770C) transition, and a deletion of nucleotides 2896-2911-whereas no mutations were detected in the sequence encoding the liver gamma subunit of phosphorylase kinase. The 1827G-->A mutation and the deletion both result in the formation of early stop codons. Investigation of DNA showed that the deletion is caused by a splice-acceptor site mutation (IVS30(-1),g-->t). Family analysis revealed that the 1827G-->A and IVS30(-1),g-->t substitutions are located on different parental chromosomes and that compound heterozygosity for these mutations segregates with the disease. The 2309A-->G mutation was detected in 2%-3% of the normal population. Thus, it is concluded that the deficiency of phosphorylase kinase in this proband is caused by compound heterozygosity for the 1827G-->A and the IVS30(-1),g-->t mutations and that the 2309A-->G mutation is a polymorphism. This implies that a defect in the sequence encoding the common part of the beta subunit of phosphorylase kinase may present as liver phosphorylase kinase deficiency.     

Prevalence of nine mutations among Jewish and non-Jewish Gaucher disease patients.

The frequency of nine different mutated alleles known to occur in the glucocerebrosidase gene was determined in 247 Gaucher patients, of whom 176 were of Jewish extraction, 2 were Jewish with one converted parent, and 69 were of non-Jewish origin. DNA was prepared from peripheral blood, active glucocerebrosidase sequences were amplified by using the PCR technique, and the mutations were identified by using the allele-specific oligonucleotide hybridization method. The N37OS mutation appeared in 69.77% of the mutated alleles in Jewish patients and in 22.86% of the mutated alleles in non-Jews. The 84GG mutation, which has not been found so far among non-Jewish patients, existed in 10.17% of the disease alleles among Jewish patients. The IVS + 1 mutation constituted 2.26% of the disease alleles among Jewish patients and 1.43% among the non-Jewish patients. RecTL, a complex allele containing four single-base-pair changes, occurred in 2.26% of the alleles in Jewish patients and was found in two (1.43%) of the patients of non-Jewish extraction. Another complex allele, designated "RecNciI" and containing three single-point mutations, appeared in 7.8% of alleles of non-Jewish patients and in only two (0.56%) of the Jewish families. The prevalence of the L444P mutation among non-Jewish Gaucher patients was 31.43%, while its prevalence among Jewish patients was only 4.24%. The prevalence of two other point mutations--D409H and R463C--was 5.00% and 3.57%, respectively, among non-Jewish patients and was not found among the Jewish Gaucher patient population. The prevalence of the R496H mutation, found so far only among Jewish patients, was 1.13%. The results presented demonstrate that seven mutations identify 90.40% of the mutations among Jewish patients and that these seven mutations allow diagnosis of only 73.52% of the non-Jewish patients. Identification of additional mutant alleles will enhance the accuracy of carrier detection.     

Identification of a frequent pseudodeficiency mutation in the fumarylacetoacetase gene, with implications for diagnosis of tyrosinemia type I.

In healthy individuals, fumarylacetoacetase (FAH) activities close to the range found in hereditary tyrosinemia type 1 (HT1) patients indicated the existence of a "pseudodeficiency" allele. In an individual homozygous for pseudodeficiency of FAH and in three HT1 families also carrying the pseudodeficiency allele, western blotting of fibroblast extracts showed that the pseudodeficiency allele gave very little immunoreactive FAH protein, whereas northern analysis revealed a normal amount of FAH mRNA. Sequencing revealed an identical mutation, C1021-->T (Arg341Trp), in all the pseudodeficiency alleles. Site-directed mutagenesis and expression in a rabbit reticulocyte lysate system demonstrated that the C1021-->T mutation gave reduced FAH activity and reduced amounts of the full-length protein. Bs1EI restriction digestion of PCR products distinguished between the normal and the mutated sequences. Among 516 healthy volunteers of Norwegian origin, the C1021-->T mutation was found in 2.2% of the alleles. Testing for the C1021-->T mutation may solve the problem of prenatal diagnosis and carrier detection in families with compound heterozygote genotypes for HT1 and pseudodeficiency.     

Segregation of Tay-Sachs and Sandhoff alleles in a non-Jewish family.

A non-Jewish family is presented in which the genes for Tay-Sachs disease and Sandhoff disease are segregating. Individuals heterozygous for both alleles have low serum and white cell total hexosaminidase levels together with a proportion of heat-labile hexosaminidase A (HEX A) which falls in the normal range. The individuals would not be detected as carriers of Tay-Sachs disease or Sandhoff disease in a population screening program.     

A second common mutation in the methylenetetrahydrofolate reductase gene: an additional risk factor for neural-tube defects?

Recently, we showed that homozygosity for the common 677(C-->T) mutation in the methylenetetrahydrofolate reductase (MTHFR) gene, causing thermolability of the enzyme, is a risk factor for neural-tube defects (NTDs). We now report on another mutation in the same gene, the 1298(A-->C) mutation, which changes a glutamate into an alanine residue. This mutation destroys an MboII recognition site and has an allele frequency of .33. This 1298(A-->C) mutation results in decreased MTHFR activity (one-way analysis of variance [ANOVA] P < .0001), which is more pronounced in the homozygous than heterozygous state. Neither the homozygous nor the heterozygous state is associated with higher plasma homocysteine (Hcy) or a lower plasma folate concentration-phenomena that are evident with homozygosity for the 677(C-->T) mutation. However, there appears to be an interaction between these two common mutations. When compared with heterozygosity for either the 677(C-->T) or 1298(A-->C) mutations, the combined heterozygosity for the 1298(A-->C) and 677(C-->T) mutations was associated with reduced MTHFR specific activity (ANOVA P < .0001), higher Hcy, and decreased plasma folate levels (ANOVA P <.03). Thus, combined heterozygosity for both MTHFR mutations results in similar features as observed in homozygotes for the 677(C-->T) mutation. This combined heterozygosity was observed in 28% (n =86) of the NTD patients compared with 20% (n =403) among controls, resulting in an odds ratio of 2.04 (95% confidence interval: .9-4.7). These data suggest that the combined heterozygosity for the two MTHFR common mutations accounts for a proportion of folate-related NTDs, which is not explained by homozygosity for the 677(C-->T) mutation, and can be an additional genetic risk factor for NTDs.     

Frequency of three Hex A mutant alleles among Jewish and non-Jewish carriers identified in a Tay-Sachs screening program.

Mutations in the HEX A gene, encoding the alpha-subunit of beta-hexosaminidase A (Hex A), are the cause of Tay-Sachs disease as well as of juvenile, chronic, and adult GM2 gangliosidoses. We have examined the distribution of three mutations--a 4-nucleotide insertion in exon 11, a G----C transversion at a 5' splice site in intron 12, and a 269Gly----Ser amino acid substitution in exon 7--among individuals enzymatically diagnosed as carriers of Hex A deficiency. Mutation analysis included polymerase chain reaction (PCR) amplification of the relevant regions of genomic DNA, followed by allele-specific oligonucleotide hybridization; another test for heterozygosity of the exon 11 insertion was based on the formation of heteroduplex PCR fragments of low electrophoretic mobility. The percentage distribution of the exon 11, intron 12, exon 7, and unidentified mutant alleles was 73:15:4:8 among 156 Jewish carriers of Hex A deficiency and 16:0:3:81 among 51 non-Jewish carriers. Regardless of the mutation, the ancestral origin of the Jewish carriers was primarily eastern and (somewhat less often) central Europe, whereas for the non-Jewish carriers it was western Europe. Because a twelfth of the Jewish carriers and four-fifths of the non-Jewish carriers of Hex A deficiency had mutant alleles other than the three common ones tested, enzyme-based tests cannot be replaced by DNA-based tests at the present time. However, DNA-based tests for two-carrier couples could identify those at risk for the chronic/adult GM2 gangliosidoses rather than for infantile Tay-Sachs disease.     

Development of a mutation hotspot detection kit for the phenylalanine hydroxylase gene by ARMS-PCR combined with fluorescent probe technology

To develop a screening kit for detecting mutation hotspots of the phenylalanine hydroxylase (PAH) gene. Thirteen exons of the PAH gene were sequenced in 84 cases with phenylketonuria (PKU) diagnosed during neonatal genetic and metabolic disease screening in Shaanxi province, and their mutations were analyzed. We designed and developed a screening kit to detect nine mutation sites covering more than 50% of the PAH mutations found in Shaanxi province (c.728G>A, c.1197A>T, c.331C>T, c.1068C>A, c.611A>G, c.1238G>C, c.721C>T, c.442-1G>A, and c.158G>A) by using amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) combined with fluorescent probe technology. Peripheral blood and dried blood samples from PKU families were used for clinical verification of the newly developed kit. PAH gene mutations were detected in 84 children diagnosed with PKU. A total of 159 mutant alleles were identified, consisting of 100 missense mutations, 28 shear mutations, 24 nonsense mutations, and 7 deletion mutations. Exon 7 had the highest mutation frequency (32.08%). Among them, the mutation frequency of p.R243Q was the highest, accounting for 20.13% of all mutations, followed by p.R111X, IVS4-1G>A, EX6-96A>G, and p.R413P; these five loci accounted for 47.17% (75/159) of all mutations. In addition, we identified three previously unreported PAH gene mutations (p.C334X, p.G46D, and p.G256D). Fifteen mutation sites were identified in the 47 PAH carriers identified by next-generation sequencing (NGS), which were verified by the newly developed kit, with an agreement rate of 100%. This newly developed kit based on ARMS-PCR combined with fluorescent probe technology can be used to detect common PAH gene mutations.     

Six novel deleterious and three neutral mutations in the gene encoding the alpha-subunit of hexosaminidase A in non-Jewish individuals.

Initial investigations demonstrated that only 3/34 "Tay-Sachs chromosomes" in 22 unrelated, non-Jewish patients or carriers of some form of GM2-gangliosidosis (7 black and 15 non-Jewish Caucasian) had either of the two mutations commonly found in the Jewish population. To determine the nature and incidence of the alterations in this non-Jewish population we have utilized PCR, single-strand conformation polymorphism analysis and sequencing to detect new mutations in genomic DNA. Fourteen primer sets have been utilized to analyze 80% of the coding region and 23/26 splice sites of the gene coding for the alpha chain of hexosaminidase A. Presumed deleterious mutations were discovered in 17/34 chromosomes believed to be carrying a beta-hexosaminidase A alpha-subunit gene mutation. Ten had abnormalities which have been described previously. In the remaining 24 Tay-Sachs disease alleles, six novel mutations predicted to be deleterious were discovered. These include two small deletions (a single-base frameshift and a three-base deletion removing an amino acid), two different nonsense mutations, an initiation codon mutation (ATG----GTG), and a missense mutation (Arg499Cys) in a highly conserved residue. In addition, three presumed nondeleterious mutations were found.     

The DYT1 Gene on 9q34 Is Responsible for Most Cases of Early Limb-Onset Idiopathic Torsion Dystonia in Non-Jews

Idiopathic torsion dystonia (ITD) is characterized by involuntary twisting movements and postures. A gene for this disorder, DYT1, was mapped to chromosome 9q34 in 12 Ashkenazi Jewish (AJ) families and one large non-Jewish kindred. In the AJ population, strong linkage disequilibrium exists between DYT1 and adjacent markers within a 2-cM region. The associated haplotype occurs in >90% of early limb-onset AJ cases. We examined seven non-Jewish ITD families of northern European and French Canadian descent to determine the extent to which early-onset ITD in non-Jews maps to DYT1. Results are consistent with linkage to the DYT1 region. Affected individuals in these families are clinically similar to the AJ cases; i.e., the site of onset is predominantly in the limbs and at least one individual in each pedigree had onset before age 12 years. None carries the AJ haplotype; therefore, they probably represent different mutations in the DYT1 gene. The two French Canadian families, however, display the same haplotype. Estimates of penetrance in non-Jewish families range from .40 to .75. We identified disease gene carriers and, with adjustments for age at onset, obtained a direct estimate of penetrance of .46. This is consistent with estimates of 30%–40% in the AJ population. Two other non-Jewish families with atypical ITD (later onset and/or cranial or cervical involvement) are not linked to DYT1, which indicates involvement of other genes in dystonia.     

Molecular genetic analysis in mild hyperhomocysteinemia: a common mutation in the methylenetetrahydrofolate reductase gene is a genetic risk factor for cardiovascular disease.

Mild hyperhomocysteinemia is an established risk factor for cardiovascular disease. Genetic aberrations in the cystathionine beta-synthase (CBS) and methylenetetrahydrofolate reductase (MTHFR) genes may account for reduced enzyme activities and elevated plasma homocysteine levels. In 15 unrelated Dutch patients with homozygous CBS deficiency, we observed the 833T-->C (I278T) mutation in 50% of the alleles. Very recently, we identified a common mutation (677C-->T; A-->V) in the MTHFR gene, which, in homozygous state, is responsible for the thermolabile phenotype and which is associated with decreased specific MTHRF activity and elevated homocysteine levels. We screened 60 cardiovascular patients and 111 controls for these two mutations, to determine whether these mutations are risk factors for premature cardiovascular disease. Heterozygosity for the 833T-->C mutation in the CBS gene was observed in one individual of the control group but was absent in patients with premature cardiovascular disease. Homozygosity for the 677C-->T mutation in the MTHFR gene was found in (15%) of 60 cardiovascular patients and in only 6 (approximately 5%) of 111 control individuals (odds ratio 3.1 [95% confidence interval 1.0-9.2]). Because of both the high prevalence of the 833T-->C mutation among homozygotes for CBS deficiency and its absence in 60 cardiovascular patients, we may conclude that heterozygosity for CBS deficiency does not appear to be involved in premature cardiovascular disease. However, a frequent homozygous mutation in the MTHFR gene is associated with a threefold increase in risk for premature cardiovascular disease.