W474C amino acid substitution affects early processing of the α-subunit of β-hexosaminidase A and is associated with subacute GM2 gangliosidosis

Mutations in the HEXA gene, encoding the α-subunit of β-hexosaminidase A (Hex A), that abolish Hex A enzyme activity cause Tay-Sachs disease (TSD), the fatal infantile form of G_M2 gangliosidosis, Type 1. Less severe, subacute (juvenile-onset) and chronic (adult-onset) variants are characterized by a broad spectrum of clinical manifestations and are associated with residual levels of Hex A enzyme activity. We identified a 1422 G→C (amino acid W474C) substitution in the first position of exon 13 of HEXA of a non-Jewish proband who manifested a subacute variant of G_M2 gangliosidosis. On the second maternally inherited allele, we identified the common infantile disease-causing 4-bp insertion, +TATC 1278, in exon 11. Pulse-chase analysis using proband fibroblasts revealed that the W474C-containing α-subunit precursor was normally synthesized, but not phosphorylated or secreted, and the mature lysosomal α-subunit was not detected. When the W474C-containing α-subunit was transiently co-expressed with the β-subunit to produce Hex A (αβ) in COS-7 cells, the mature α-subunit was present, but its level was much lower than that from normal α-subunit transfections, although higher than in those cells transfected with an α-subunit associated with infantile TSD. Furthermore, the precursor level of the W474C α-subunit was found to accumulate in comparison to the normal α-subunit precursor levels. We conclude that the 1422 G→C mutation is the cause of Hex A enzyme deficiency in the proband. The resulting W474C substitution clearly interferes with α-subunit processing, but because the base substitution falls at the first position of exon 13, aberrant splicing may also contribute to Hex A deficiency in this proband. Hum Mutat 11:432–442, 1998. © 1998 Wiley-Liss, Inc.     

GM2 gangliosidosis associated with a HEXA missense mutation in Japanese Chin dogs: A potential model for Tay Sachs disease

GM2 gangliosidosis is a fatal lysosomal storage disease caused by a deficiency of β-hexosaminidase (EC 3.2.1.52). There are two major isoforms of the enzyme: hexosaminidase A composed of an α and a β subunit (encoded by HEXA and HEXB genes, respectively); and, hexosaminidase B composed of two β subunits. Hexosaminidase A requires an activator protein encoded by GM2A to catabolize GM2 ganglioside, but even in the absence of the activator protein, it can hydrolyze the synthetic substrates commonly used to assess enzyme activity. GM2 gangliosidosis has been reported in Japanese Chin dogs, and we identified the disease in two related Japanese Chin dogs based on clinical signs, histopathology and elevated brain GM2 gangliosides. As in previous reports, we found normal or elevated hexosaminidase activity when measured with the synthetic substrates. This suggested that the canine disease is analogous to human AB variant of G_M2 gangliosidosis, which results from mutations in GM2A. However, only common neutral single nucleotide polymorphisms were found upon sequence analysis of the canine ortholog of GM2A from the affected Japanese Chins. When the same DNA samples were used to sequence HEXA, we identified a homozygous HEXA:c967G>A transition which predicts a p.E323K substitution. The glutamyl moiety at 323 is known to make an essential contribution to the active site of hexosaminidase A, and none of the 128 normal Japanese Chins and 92 normal dogs of other breeds that we tested was homozygous for HEXA:c967A. Thus it appears that the HEXA:c967G>A transition is responsible for the GM2 gangliosidosis in Japanese Chins.     

Molecular basis of heat labile hexosaminidase B among Jews and Arabs

Genotyping individuals for Tay-Sachs disease (TSD) is mainly based on the heat lability of β-hexosaminidase (Hex) A (αβ) and the heat stability of Hex B (ββ). Mutations in the HEXB gene encoding the β-subunits of Hex that result in heat-labile Hex B thus may lead to erroneous enzymatic genotyping regarding TSD. Utilizing single strand conformation polymorphism (SSCP) analysis for all 14 exons of HEXB followed by direct sequencing of aberrant fragments, we screened individuals whose Hex B was heat labile. A novel heat labile mutation, previously concluded to exist in the HEXB gene, was identified among Jews and Arabs as 1627 G→A. One individual with heat labile Hex B (HLB) was negative for this novel mutation and for the known 1514 G→A HLB mutation, proving that there exists at least one other unidentified HLB mutation. Based on these results, it is advisable to perform DNA tests for 1627 G→A mutation in suspected HLB individuals. Hum Mutat 10:424–429, 1997. © 1997 Wiley-Liss, Inc.     

Inborn errors of lysosomal catabolism—principles of heterozygote detection

Carriers of an inborn error of lysosomal catabolism can be recognized, as they have enzyme levels approximately half those of normal individuals. Of the various tissues readily available for assay, plasma and leuk ocytes and, in some situations, tears are preferred. Although mixed leuk ocytes have proved satisfactory in Tay-Sachs screening programs, purified preparations of granulocytes or lymphocytes will allow better discrimination in most situations. Enzymes are assayed relative to some other reference parameter which must be a constant or highly correlated with test enzyme activity. In the two mass screening programs in operation, β-hexosaminidase A and α-mannosidase have both been assayed relative to total β-hexosaminidase activity. Carrier detection is particularly important in X-linked diseases. The techniques used mostly involve hair roots or fibroblasts and depend on random inactivation of the X chromosome. In the mucolipid oses II and III, in which there are a number of deficient enzymes in cells, carriers may be identified on the basis of the ratio of β-hexosaminidase I₁ and I₂ to total hexosaminidase.     

Tay-Sachs disease and HEXA mutations among Moroccan Jews

Moroccan Jewry (N>750,000) is the only non-Ashkenazi Jewish community in which Tay-Sachs disease (TSD) is not extremely rare. Previous studies among Moroccan Jewish TSD families identified three HEXA mutations. In this study, extended to enzyme-defined and new obilgate TSD carriers, we found four additional mutations. One of them is a novel, IVS5-2(A→G) substitution, resulting in exon skipping, and it was found only among enzyme-defined carriers. The seven HEXA identified mutations among Moroccan Jews are: ΔF^304/305, R170Q, IVS-2(A→G), Y180X, E482K, 1278+TATC, and IVS12+1(G→C). Their respective distribution among 51 unrelated enzyme-defined and obligate carriers is 22:19:6:1:1:1:1. The mutation(s) remain unknown in only three enzyme-defined carriers. Five of the seven Moroccan mutations, including the three most common ones, were not found among Ashkenazi Jews. Compared with the much larger and relatively homogeneous Ashkenazi population, the finding among Moroccan Jews probably reflects their much longer history. Hum Mutat 10:295–300, 1997. © 1997 Wiley-Liss, Inc.     

Comparison of enzyme and DNA analysis in a Tay-Sachs disease carrier screening program.

Tay-Sachs disease (GM2 gangliosidosis, type 1; TSD) is an autosomal recessive GM2 gangliosidosis resulting from the deficient activity of the lysosomal hydrolase beta-hexosaminidase A (Hex A). With a carrier frequency estimated at 1 in 25, it is a common lysosomal disorder in the Ashkenazi Jewish population. Tay-Sachs disease has provided the prototype for the prevention of severe recessive genetic diseases. Molecular analysis of the Hex A gene (HEXA) of Ashkenazi Jewish individuals affected with Tay-Sachs disease revealed that three common mutations cause the infantile and adult onset forms of the disease; a four base insertion in exon 11, a splice junction mutation in intron 12 and a point mutation in exon 7 (G269S). A study was undertaken to determine whether mutation analysis would be useful in TSD screening programs in identifying carriers and clarifying the status of individuals whose enzyme assays are inconclusive. Ashkenazi Jewish individuals who had been diagnosed as carriers, inconclusives by enzyme assay and non-carriers with low normal enzyme levels in the Mount Sinai Tay-Sachs Disease Prevention Program were examined for the presence of the three mutations using polymerase chain reaction (PCR) and allele specific oligonucleotide (ASO) hybridization. The insertion mutation was present in 29 of 34 carriers and 2 of 36 inconclusive individuals, the splice junction mutation was found in 4 of 34 carriers and the G269S mutation was found in 1 of 34 carriers. Of the 313 non-carrier individuals with normal enzyme activity in the lower normal range, one was positive for the splice junction mutation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tay-Sachs disease in Jacob sheep

Autopsy studies of four Jacob sheep dying within their first 6–8 months of a progressive neurodegenerative disorder suggested the presence of a neuronal storage disease. Lysosomal enzyme studies of brain and liver from an affected animal revealed diminished activity of hexosaminidase A (Hex A) measured with an artificial substrate specific for this component of β-hexosaminidase. Absence of Hex A activity was confirmed by cellulose acetate electrophoresis. Brain lipid analyses demonstrated the presence of increased concentrations of G_M2-ganglioside and asialo-G_M2-ganglioside. The hexa cDNA of Jacob sheep was cloned and sequenced revealing an identical number of nucleotides and exons as in human HexA and 86% homology in nucleotide sequence. A missense mutation was found in the hexa cDNA of the affected sheep caused by a single nucleotide change at the end of exon 11 resulting in skipping of exon 11. Transfection of normal sheep hexa cDNA into COS1 cells and human Hex A-deficient cells led to expression of Hex S but no increase in Hex A indicating absence of cross-species dimerization of sheep Hex α-subunit with human Hex β-subunits. Using restriction site analysis, the heterozygote frequency of this mutation in Jacob sheep was determined in three geographically separate flocks to average 14%. This large naturally occurring animal model of Tay-Sachs disease is the first to offer promise as a means for trials of gene therapy applicable to human infants.     

Identification of two HEXA mutations causing infantile-onset Tay-Sachs disease in the Persian population

The β-hexosaminidase A (HEXA) mutations in the first reported cases of infantile Tay-Sachs disease in the Persian population were identified in two unrelated consanguineous families. The clinical diagnoses of the affected infants were confirmed by their markedly deficient levels of HEXA activity in plasma or peripheral leukocytes. The specific causative mutation in each family was determined by sequencing the HEXA alleles in both sets of related parents. Two mutations were identified: c.1A>G (p.MIV), which obliterated the initiating methionine in codon 1, and c.1177C>T (p.R393X), which predicted a termination codon or nonsense mutation.     

Population-specific screening by mutation analysis for diseases frequent in Ashkenazi Jews

We describe a partially automated DNA mutation assay for detecting the most frequent mutations in the α-subunit of β-hexosaminidase A, the acid β-glucosidase and the cystic fibrosis transmembrane conductance regulator genes for the Ashkenazi Jewish population. The assay detects carriers for Tay-Sachs disease, Gaucher disease, and cystic fibrosis with sensitivities of at least 92%, 96%, and 97%, respectively. Among 1,364 young adults of Ashkenazic ancestry in the Dor Yeshurim community who were tested, 52 were Tay-Sachs carriers, 110 were Gaucher carriers, and 62 were cystic fibrosis carriers. Ten individuals were carriers for two diseases, and three unsuspected cases were diagnosed with Gaucher disease based on mutation test results. In addition to Tay-Sachs mutation data, results for hexosaminidase A activity were also available. All of 1,254 samples normal by enzyme quantitation were also negative for the three α-subunit mutations tested, and all of 43 samples with ‘inconclusive’ enzyme results were negative by DNA. Only 52 of 67 samples positive by enzyme assay were also positive for one of the three mutations tested for Tay-Sachs disease. The data suggest a high degree of false positivity inherent in enzyme identification of carriers. There are no correlative methods to assess the sensitivity of Gaucher and CF carrier testing. The results show that population screening can be carried out efficiently by DNA analysis, with the accrual of carrier information for three separate diseases conducted as a single test. Furthermore, the DNA method for Tay-Sachs screening appears to exceed the specificity of hexosaminidase A enzyme testing. © 1996 Wiley-Liss, Inc.     

Biochemical evaluation of intracerebroventricular rhNAGLU-IGF2 enzyme replacement therapy in neonatal mice with Sanfilippo B syndrome

Mucopolysaccharidosis IIIB (MPS IIIB, Sanfilippo syndrome type B) is caused by a deficiency in α-N-acetylglucosaminidase (NAGLU) activity, which leads to the accumulation of heparan sulfate (HS). MPS IIIB causes progressive neurological decline, with affected patients having an expected lifespan of approximately 20 years. No effective treatment is available. Recent pre-clinical studies have shown that intracerebroventricular (ICV) ERT with a fusion protein of rhNAGLU-IGF2 is a feasible treatment for MPS IIIB in both canine and mouse models. In this study, we evaluated the biochemical efficacy of a single dose of rhNAGLU-IGF2 via ICV-ERT in brain and liver tissue from Naglu^?/? neonatal mice. Twelve weeks after treatment, NAGLU activity levels in brain were 0.75-fold those of controls. HS and β-hexosaminidase activity, which are elevated in MPS IIIB, decreased to normal levels. This effect persisted for at least 4 weeks after treatment. Elevated NAGLU and reduced β-hexosaminidase activity levels were detected in liver; these effects persisted for up to 4 weeks after treatment. The overall therapeutic effects of single dose ICV-ERT with rhNAGLU-IGF2 in Naglu^?/? neonatal mice were long-lasting. These results suggest a potential benefit of early treatment, followed by less-frequent ICV-ERT dosing, in patients diagnosed with MPS IIIB.     

Effectiveness of plasma lyso-Gb3 as a biomarker for selecting high-risk patients with Fabry disease from multispecialty clinics for genetic analysis

PurposePlasma globotriaosylsphingosine (lyso-Gb3) is a promising secondary screening biomarker for Fabry disease. Here, we examined its applicability as a primary screening biomarker for classic and late-onset Fabry disease in males and females.MethodsBetween 1 July 2014 and 31 December 2015, we screened 2,360 patients (1,324 males) referred from 169 Japanese specialty clinics (cardiology, nephrology, neurology, and pediatrics), based on clinical symptoms suggestive of Fabry disease. We used the plasma lyso-Gb3 concentration, α-galactosidase A (α-Gal A) activity, and analysis of the α-Gal A gene (GLA) for primary and secondary screens, respectively.ResultsOf 8 males with elevated lyso-Gb3 levels (≥2.0 ng ml^–1) and low α-Gal A activity (≤4.0 nmol h^–1 ml^–1), 7 presented a GLA mutation (2 classic and 5 late-onset). Of 15 females with elevated lyso-Gb3, 7 displayed low α-Gal A activity (5 with GLA mutations; 4 classic and 1 late-onset) and 8 exhibited normal α-Gal A activity (1 with a classic GLA mutation and 3 with genetic variants of uncertain significance).ConclusionPlasma lyso-Gb3 is a potential primary screening biomarker for classic and late-onset Fabry disease probands.     

Duplication of amyloid precursor protein (APP), but not prion protein (PRNP) gene is a significant cause of early onset dementia in a large UK series

Amyloid precursor protein gene (APP) duplications have been identified in screens of selected probands with early onset familial Alzheimer's disease (FAD). A causal role for copy number variation (CNV) in the prion protein gene (PRNP) in prion dementias is not known. We aimed to determine the prevalence of copy number variation in APP and PRNP in a large referral series, test a screening method for detection of the same, and expand knowledge of clinical phenotype. We used a 3-tiered screening assay for APP and PRNP duplication (exonic real-time quantitative polymerase chain reaction [exon-qPCR], fluorescent microsatellite quantitative PCR [fm-q-PCR], and Illumina array [Illumina Inc., San Diego, CA, USA]) for analysis of a heterogeneous referral series comprising 1531 probands. Five of 1531 probands screened showed APP duplication, a similar prevalence to APP missense mutation. Real-time quantitative PCR and fluorescent microsatellite quantitative PCR were similar individually but are theoretically complementary; we used Illumina arrays as our reference assay. Two of 5 probands were from an autosomal dominant early onset Alzheimer's disease (familial Alzheimer's disease) pedigree. One extensive, noncontiguous duplication on chromosome 21 was consistent with an unbalanced translocation not including the Down's syndrome critical region. Seizures were prominent in the other typical APP duplications. A range of imaging, neuropsychological, cerebrospinal fluid, and pathological findings are reported that extend the known phenotype. APP but not PRNP duplication is a significant cause of early onset dementia in the UK. The recognized phenotype may be expanded to include the possibility of early seizures and apparently sporadic disease which, in part, may be due to different mutational mechanisms. The pros and cons of our screening method are discussed.     

Heterozygosity for Tay-Sachs disease in non-Jewish Americans with ancestry from Ireland or Great Britain.

We performed a genetic epidemiological analysis of American non-Jewish people with ancestry from Ireland or Great Britain with regard to heterozgosity for Tay-Sachs disease (TSD). This study was prompted by a recent report that the frequency of heterozygosity for TSD among Irish Americans was 1 in 8, a frequency much higher than that recognised for any other population group. We identified 19 of 576 (3.3%) people of Irish background as TSD heterozygotes by the standard thermolability assay for beta-hexosaminidase A (Hex A) activity. Three of 289 people of non-Irish British Isles background (1%) were also identified as heterozygotes by biochemical testing. Specimens from the biochemically identified Irish heterozygotes were analysed for seven different Hex A alpha subunit gene mutations; three (15.8%) had a lethal +1 IVS-9 G to A mutation, previously noted to be a common mutation among TSD heterozygotes of Irish ancestry. Eight of 19 (42.1%) had one of two benign or pseudodeficiency mutations, and no mutation was found in 42.1% of the heterozygotes analysed. These data indicate that non-Jewish Americans with Irish background have a significantly increased frequency of heterozygosity at the Hex A alpha subunit gene locus, but that approximately 42% of the biochemically ascertained heterozygotes have clinically benign mutations. A pseudodeficiency mutation was identified in one of the three TSD heterozygotes of non-Irish British Isles background; no mutations were found in the other two. The data allow for a frequency estimate of deleterious alleles for TSD among Irish Americans of 1 in 192 to 1 in 52. Non-Jewish Americans with ancestry from Great Britain have a minimal, if any, increase in rate of heterozygosity at the TSD gene locus relative to the general population.     

De novo recurrent germline mutation of the BRCA2 gene in a patient with early onset breast cancer

Germline mutations in either of the two major breast cancer predisposition genes, BRCA1 and BRCA2, account for a significant proportion of hereditary breast/ovarian cancer. Identification of breast cancer patients carrying mutations of these genes is primarily based on a positive family history of breast/ovarian cancer or early onset of the disease or both. In the course of mutation screening of the BRCA1 and BRCA2 genes in a hospital based series of patients with risk factors for hereditary breast/ovarian cancer, we identified a germline mutation in the BRCA2 gene (3034del4) in a patient with early onset breast cancer and no strong family history of the disease. Subsequent molecular analysis in her parents showed that neither of them carried the mutation. Paternity was confirmed using a set of highly polymorphic markers, showing that the proband carried a de novo germline mutation in the BRCA2 gene. Interestingly, 3034del4 is a recurrent mutation occurring in a putative mutation prone region of the BRCA2 gene. Our study presents the first case in which a de novo germline mutation in the BRCA2 gene has been identified, and supports previous results of haplotype studies, confirming that the 3034del4 mutation has multiple independent origins.


Keywords: breast cancer; BRCA2 gene; de novo mutation     

Lysosomal storage disease in the brain: mutations of the β-mannosidase gene identified in autosomal dominant nystagmus

PurposeGenetic etiology of congenital/infantile nystagmus remains largely unknown. This study aimed to identify genomic mutations in patients with infantile nystagmus and an associated disease network.MethodsPatients with inherited and sporadic infantile nystagmus were recruited for whole-exome and Sanger sequencing. β-Mannosidase activities were measured. Gene expression, protein–protein interaction, and nystagmus-associated lysosomal storage disease (LSD) genes were analyzed.ResultsA novel heterozygous mutation (c.2013G>A; p.R638H) of MANBA, which encodes lysosomal β-mannosidase, was identified in patients with autosomal-dominant nystagmus. An additional mutation (c.2346T>A; p.L749H) in MANBA was found by screening patients with sporadic nystagmus. MANBA was expressed in the pretectal nucleus of the developing midbrain, known to be involved in oculomotor and optokinetic nystagmus. Functional validation of these mutations demonstrated a significant decrease of β-mannosidase activities in the patients as well as in mutant-transfected HEK293T cells. Further analysis revealed that nystagmus is present in at least 24 different LSDs involving the brain.ConclusionThis is the first identification of MANBA mutations in patients with autosomal-dominant nystagmus, suggesting a new clinical entity. Because β-mannosidase activities are required for development of the oculomotor nervous system, our findings shed new light on the role of LSD-associated genes in the pathogenesis of infantile nystagmus.     

SOD1 G93D sporadic amyotrophic lateral sclerosis (SALS) patient with rapid progression and concomitant novel ANG variant

SOD1 G93D mutation has been described in amyotrophic lateral sclerosis (ALS) patients with slowly progressive disease. We describe an Italian patient affected by sporadic ALS with the SOD1 G93D mutation that disclosed an unusual rapid progression with death occurring after 30 months from the symptom onset. Considering the atypical clinical course further genes associated with ALS or known to be causative were studied including ANG, PGRN, TARDBP, FUS, VCP, CHRNA3, CHRNA4, and CHRNB4. A novel heterozygous ANG missense variant (c.433 C>T, p.R145C) was identified which is neither reported in controls nor in 1000 genomes and single nucleotide polymorphism (SNP) databases. This report confirms that clinical course of SOD1-related ALS may be modulated by other causative or associated genes, including ANG and suggests that extensive screening of ALS-associated genes in patients with an already identified mutation may be helpful for better knowledge of genetic architecture of ALS.     

Juvenile-onset motor neuron disease caused by novel mutations in β-hexosaminidase

A 12 year-old female presented with a seven-year history of progressive muscle weakness, atrophy, tremor and fasciculations. Cognition was normal. Rectal biopsy revealed intracellular storage material and biochemical testing indicated low hexosaminidase activity consistent with juvenile-onset G_M2-gangliosidosis. Genetic evaluation revealed compound heterozygosity with two novel mutations in the hexosaminidase β-subunit (c.512-3 C>A and c.1613+15_1613+18dup). Protein analysis was consistent with biochemical findings and indicated only a small portion of β-subunits were properly processed. These results provide additional insight into juvenile-onset G_M2-gangliosidoses and further expand the number of β-hexosaminidase mutations associated with motor neuron disease.     

Mutational analysis in early-onset familial Alzheimer's disease in Mainland China

Mutations of 3 causative genes, namely presenilin 1 (PSEN1), presenilin 2 (PSEN2), and amyloid precursor protein (APP), have been identified as the major causes of early-onset familial Alzheimer's disease (EOFAD). Recently, a GGGGCC repeat expansion in the noncoding region of C9orf72 was also detected in some patients with clinically diagnosed familial Alzheimer's disease. The prevalence of causative gene mutations in patients with EOFAD has been reported in previous studies but their prevalence remains unclear in Mainland China. The aim of this study was to characterize the common causative gene mutation spectrum and genotype-phenotype correlations in Chinese patients with EOFAD. Genetic screening for mutations in PSEN1, PSEN2, and APP was conducted in a total of 32 families with clinical diagnoses of EOFAD from Mainland China. Subsequently, a hexanucleotide repeat expansion in C9orf72 was detected in all patients. Four novel mutations in PSEN1 (p.A434T, p.I167del, p.F105C, and p.L248P) were identified in 4 respective families, and 1 previously recognized pathogenic mutation in APP (p.V717I) was detected in another 2 unrelated families. The PSEN2 mutation and pathogenic repeat expansions of C9orf72 were not detected in all patients. To the best of our knowledge, this is the first cohort report of a causative gene screen in patients with EOFAD in Mainland China. The analysis of the genetic-clinical correlations in this cohort supports the idea that the clinical phenotype might be influenced by specific genetic defects.