Identification of mutations in the ALD-gene of 20 families with adrenoleukodystrophy/adrenomyeloneuropathy

Adrenoleukodystrophy (ALD), an X-linked inherited metabolic disorder, is the most frequent inborn peroxisomal disease. It leads to demyelination in the central and peripheral nervous system. Defective -oxidation of saturated very long chain fatty acids (VLCFAs; C22:0–C26:0) in peroxisomes has been shown to lead to an accumulation of VLCFAs in leukoid areas of the central nervous system, peripheral nerves, adrenal gland, and blood. The ALD gene has been recently identified and encodes a 745-amino-acid protein. We screened patients with adrenoleukodystrophy/adrenomyeloneuropathy (ALD/AMN) from 20 kindreds for mutations in the ALD gene. Eleven missense and two nonsense mutations, five deletions, and one insertion were detected by direct sequencing of eight reverse transcribed fragments of the ALD-gene mRNA. Four mutations could be shown to be de novo. All mutations could be confirmed in carriers by sequencing genomic DNA. No correlation between the type of mutation and the severity of the phenotype could be observed. The mutations were not detected in the ALD gene of 30 healthy persons.     

De novo missense mutation Y174S in exon 1 of the adrenoleukodystrophy (ALD) gene

Adrenoleukodystrophy (ALD) is an X-linked disease, characterised by an alteration of the peroxisomal -oxidation of the very long chain fatty acids. The ALD gene has been identified and mutations have been detected in ALD patients. We report here a new missense mutation in the ALD gene of a male patient, predicting a tyrosine to serine substitution at codon 174 (mutation Y174S). The mother of the ALD patient does not have the Y174S mutation in her leukocyte DNA, indicating that Y174S arose de novo in the patient. Y174S is the first reported de novo mutation in the ALD gene.     

Adrenoleukodystrophy: subcellular localization and degradation of adrenoleukodystrophy protein (ALDP/ABCD1) with naturally occurring missense mutations

Mutation in the X-chromosomal adrenoleukodystrophy gene (ALD; ABCD1) leads to X-linked adrenoleukodystrophy (X-ALD), a severe neurodegenerative disorder. The encoded adrenoleukodystrophy protein (ALDP/ABCD1) is a half-size peroxisomal ATP-binding cassette protein of 745 amino acids in humans. In this study, we chose nine arbitrary mutant human ALDP forms (R104C, G116R, Y174C, S342P, Q544R, S606P, S606L, R617H, and H667D) with naturally occurring missense mutations and examined the intracellular behavior. When expressed in X-ALD fibroblasts lacking ALDP, the expression level of mutant His-ALDPs (S606L, R617H, and H667D) was lower than that of wild type and other mutant ALDPs. Furthermore, mutant ALDP-green fluorescence proteins (S606L and H667D) stably expressed in CHO cells were not detected due to rapid degradation. Interestingly, the wild type ALDP co-expressed in these cells also disappeared. In the case of X-ALD fibroblasts from an ALD patient (R617H), the mutant ALDP was not detected in the cells, but appeared upon incubation with a proteasome inhibitor. When CHO cells expressing mutant ALDP-green fluorescence protein (H667D) were cultured in the presence of a proteasome inhibitor, both the mutant and wild type ALDP reappeared. In addition, mutant His-ALDP (Y174C), which has a mutation between transmembrane domain 2 and 3, did not exhibit peroxisomal localization by immunofluorescense study. These results suggest that mutant ALDPs, which have a mutation in the COOH-terminal half of ALDP, including S606L, R617H, and H667D, were degraded by proteasomes after dimerization. Further, the region between transmembrane domain 2 and 3 is important for the targeting of ALDP to the peroxisome.     

X-Linked Adrenoleukodystrophy: Molecular and Functional Analysis of the ABCD1 Gene in Argentinean Patients

X-linked adrenoleukodystrophy (X-ALD) is an inherited metabolic disease associated with mutations in the ABCD1 gene that encodes an ATP-binding cassette transporter protein, ALDP. The disease is characterized by increased concentrations of very long-chain fatty acids (VLCFAs) in plasma and in adrenal, testicular and nervous tissues, due to a defect in peroxisomal VLCFA β-oxidation. In the present study, we analyzed 10 male patients and 17 female carriers from 10 unrelated pedigrees with X-ALD from Argentina. By sequencing the ABCD1 we detected 9 different mutations, 8 of which were novel. These new mutations were verified by a combination of methods that included both functional (western blot and peroxisomal VLCFA β-oxidation) and bioinformatics analysis. The spectrum of novel mutations consists of 3 frameshift (p.Ser284fs*16, p.Glu380Argfs*21 and p.Thr254Argfs*82); a deletion (p.Ser572_Asp575del); a splicing mutation (c.1081+5G>C) and 3 missense mutations (p.Ala341Asp, p.His420Pro and p.Tyr547Cys). In one patient 2 changes were found: a known missense (p.His669Arg) and an unpublished amino acid substitution (p.Ala19Ser). In vitro studies suggest that p.Ala19Ser is a polymorphism. Moreover, we identified two novel intronic polymorphisms and two amino acid polymorphisms. In conclusion, this study extends the spectrum of mutation in X-ALD and facilitates the identification of heterozygous females.     

Identification and expression of eight novel mutations among non-Jewish patients with Canavan disease.

Canavan disease is inherited as an autosomal recessive trait that is caused by the deficiency of aspartoacylase (ASPA). The majority of patients with Canavan disease are from an Ashkenazi Jewish background. Mutations in ASPA that lead to loss of enzymatic activity have been identified, and E285A and Y231X are the two predominant mutations that account for 97% of the mutant chromosomes in Ashkenazi Jewish patients. The current study was aimed at finding the molecular basis of Canavan disease in 25 independent patients of non-Jewish background. Eight novel and three previously characterized mutations accounted for 80% (40/50) of mutant chromosomes. The A305E missense mutation accounted for 48% (24/50) of mutant chromosomes in patients of western European descent, while the two predominant Jewish mutations each accounted for a single mutant chromosome. The eight novel mutations identified included 1- and 4-bp deletions (32 deltaT and 876 deltaAGAA, respectively) and I16T, G27R, D114E, G123E, C152Y, and R168C missense mutations. The homozygous 32 deltaT deletion was identified in the only known patient of African-American origin with Canavan disease. The heterozygosity for 876 deltaAGAA mutation was identified in three independent patients from England. Six single-base changes leading to missense mutations were identified in patients from Turkey (D114E, R168C), The Netherlands (I16T), Germany (G27R), Ireland (C152Y), and Canada (G123E). A PCR-based protocol is described that was used to introduce mutations in wild-type cDNA. In vitro expression of mutant cDNA clones demonstrated that all of these mutations led to a deficiency of ASPA and should therefore result in Canavan disease.     

Mutational and protein analysis of patients and heterozygous women with X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (ALD), a neurodegenerative disorder associated with impaired beta-oxidation of very-long-chain fatty acids (VLCFA), is due to mutations in a gene encoding a peroxisomal ATP-binding cassette (ABC) transporter (ALD protein [ALDP]). We analyzed the open reading frame of the ALD gene in 44 French ALD kindred by using SSCP or denaturing gradient-gel electrophoresis and studied the effect of mutations on ALDP by immunocytofluorescence and western blotting of fibroblasts and/or white blood cells. Mutations were detected in 37 of 44 kindreds and were distributed over the whole protein-coding region, with the exception of the C terminus encoded in exon 10. Except for two mutations (delAG1801 and P560L) observed four times each, nearly every ALD family has a different mutation. Twenty-four of 37 mutations were missense mutations leading to amino acid changes located in or close to putative transmembrane segments (TMS 2, 3, 4, and 5), in the EAA-like motif and in the nucleotide fold of the ATP-binding domain of ALDP. Of 38 ALD patients tested, 27 (71%) lacked ALDP immunoreactivity in their fibroblasts and/or white blood cells. More than half of missense mutations studied (11 of 21) resulted in a complete lack of ALDP immunoreactivity, and six missense mutations resulted in decreased ALDP expression. The fibroblasts and/or white blood cells of 15 of 15 heterozygous carrier from ALD kindred with no ALDP showed a mixture of positive- and negative-ALDP immunoreactivity due to X-inactivation. Since 5%-15% of heterozygous women have normal VLCFA levels, the immunodetection of ALDP in white blood cells can be applicable in a majority of ALD kindred, to identify heterozygous women, particularly when the ALD gene mutation has not yet been identified.     

Altered expression of ALDP in X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (ALD) is a neurodegenerative disorder with variable phenotypic expression that is characterized by elevated plasma and tissue levels of very long-chain fatty acids. However, the product of the gene defective in ALD (ALDP) is a membrane transporter of the ATP-binding cassette family of proteins and is not related to enzymes known to activate or oxidize fatty acids. We generated an antibody that specifically recognizes the C-terminal 18 amino acids of ALDP and can detect ALDP by indirect immunofluorescence. To better understand the mechanism by which mutations in ALDP lead to disease, we used this antibody to examine the subcellular distribution and relative abundance of ALDP in skin fibroblasts from normal individuals and ALD patients. Punctate immunoreactive material typical of fibroblast peroxisomes was observed in cells from seven normal controls and eight non-ALD patients. Of 35 ALD patients tested, 17 had the childhood-onset cerebral form of the disease, 13 had the milder adult phenotype adrenomyeloneuropathy, 3 had adrenal insufficiency only, and 2 were affected fetuses. More than two-thirds (69%) of all patients studied showed no punctate immunoreactive material. There was no correlation between the immunofluorescence pattern and clinical phenotype. We determined the mutation in the ALD gene in 15 of these patients. Patients with either a deletion or frameshift mutation lacked ALDP immunoreactivity, as expected. Four of 11 patients with missense mutations were also immunonegative, indicating that these mutations affected the stability or localization of ALDP. In the seven immunopositive patients with missense mutations, correlation of the location and nature of the amino acid substitution may provide new insights into the function of this peroxisomal membrane protein. Furthermore, the study of female relatives of immunonegative ALD probands may aid in the assessment of heterozygote status.     

Mutation and polymorphism analysis of the TRKA (NTRK1) gene encoding a high-affinity receptor for nerve growth factor in congenital insensitivity to pain with anhidrosis (CIPA) families

The human TRKA gene encodes a high-affinity tyrosine kinase receptor for nerve growth factor. Congenital insensitivity to pain with anhidrosis (CIPA) is an autosomal recessive genetic disorder reported from various countries and characterized by anhidrosis (inability to sweat), the absence of reaction to noxious stimuli, and mental retardation. We have found that TRKA is the gene responsible for CIPA. We have studied TRKA in 46 CIPA chromosomes derived from 23 unrelated Japanese CIPA families. including three that have been previously reported, and identified 11 novel mutations. Four (L93P, G516R, R648 C, and D668Y) are missense mutations that result in amino acid substitutions at positions conserved in the TRK family, including TRKA, TRKB, and TRKC. Three (S131 fs, L579 fs, and D770 fs) are frameshift mutations. Three (E164X, Y359X, and R596X) are nonsense mutations. The other is an intronic branch-site (IVS7-33T-->A) mutation, causing aberrant splicing in vitro. We also report the characterization of eight intragenic polymorphic sites, including a variable dinucleotide repeat and seven single nucleotide polymorphisms, and describe the haplotypic associations of alleles at these sites in 106 normal chromosomes and 46 CIPA chromosomes. More than 50% of CIPA chromosomes share the frameshift mutation (R548 fs) that we described earlier. This mutation apparently shows linkage disequilibrium with a rare haplotype in normal chromosomes, strongly suggesting that it is a common founder mutation. These findings represent the first extensive analysis of CIPA mutations and associated intragenic polymorphisms; they should facilitate the detection of CIPA mutations and aid in the diagnosis and genetic counseling of this painless but severe genetic disorder with devastating complications.     

The phenotypic spectrum of GLI3 morphopathies includes autosomal dominant preaxial polydactyly type-IV and postaxial polydactyly type-A/B; No phenotype prediction from the position of GLI3 mutations.

Functional characterization of a gene often requires the discovery of the full spectrum of its associated phenotypes. Mutations in the human GLI3 gene have been identified in Greig cepalopolysyndactyly, Pallister-Hall syndrome (PHS), and postaxial polydactyly type-A (PAP-A). We studied the involvement of GLI3 in additional phenotypes of digital abnormalities in one family (UR003) with preaxial polydactyly type-IV (PPD-IV), three families (UR014, UR015, and UR016) with dominant PAP-A/B (with PPD-A and -B in the same family), and one family with PHS. Linkage analysis showed no recombination with GLI3-linked polymorphisms. Family UR003 had a 1-nt frameshift insertion, resulting in a truncated protein of 1,245 amino acids. A frameshift mutation due to a 1-nt deletion was found in family UR014, resulting in a truncated protein of 1,280 amino acids. Family UR015 had a nonsense mutation, R643X, and family UR016 had a missense mutation, G727R, in a highly conserved amino acid of domain 3. The patient with PHS had a nonsense mutation, E1147X. These results add two phenotypes to the phenotypic spectrum caused by GLI3 mutations: the combined PAP-A/B and PPD-IV. These mutations do not support the suggested association between the mutations in GLI3 and the resulting phenotypes. We propose that all phenotypes associated with GLI3 mutations be called "GLI3 morphopathies," since the phenotypic borders of the resulting syndromes are not well defined and there is no apparent genotype-phenotype correlation.     

X-linked adrenomyeloneuropathy associated with 14 novel ALD-gene mutations: no correlation between type of mutation and age of onset

Adrenomyeloneuropathy (AMN) represents a milder form of X-linked adrenoleukodystrophy (ALD), the most frequent peroxisomal disorder. The disease is characterised by an abnormal accumulation of saturated, very long chain, fatty acids, because of altered peroxisomal ?-oxidation that concomitantly leads to demyelination in the central and peripheral nervous systems. ALD shows a highly variable phenotypic expression and extensive mutation analysis in ALD patients has failed to establish a genotype-phenotype correlation, even in the presence of the same ALD-gene defect. Therefore, we have looked for a relationship between the molecular lesion and the age of onset in 19 patients with a well-classified clinical course of AMN. The nearly complete novel spectrum of ALD gene mutations identified has revealed no obvious correlation between the type of mutation and age of AMN onset in this small series. However, intrafamiliar concordance could be observed with respect to the occurrence of adrenocortical insufficiency. This supports the idea of one (or more) additional gene(s) contributing to the phenotypic expression of ALD. Electronic Publication     

The founder mutation MSH2*1906G-->C is an important cause of hereditary nonpolyposis colorectal cancer in the Ashkenazi Jewish population

Hereditary nonpolyposis colorectal cancer (HNPCC) is caused by mutations in the mismatch-repair genes. We report here the identification and characterization of a founder mutation in MSH2 in the Ashkenazi Jewish population. We identified a nucleotide substitution, MSH2*1906G-->C, which results in a substitution of proline for alanine at codon 636 in the MSH2 protein. This allele was identified in 15 unrelated Ashkenazi Jewish families with HNPCC, most of which meet the Amsterdam criteria. Genotype analysis of 18 polymorphic loci within and flanking MSH2 suggested a single origin for the mutation. All colorectal cancers tested showed microsatellite instability and absence of MSH2 protein, by immunohistochemical analysis. In an analysis of a population-based incident series of 686 Ashkenazi Jews from Israel who have colorectal cancer, we identified 3 (0.44%) mutation carriers. Persons with a family history of colorectal or endometrial cancer were more likely to carry the mutation than were those without such a family history (P=.042), and those with colorectal cancer who carried the mutation were, on average, younger than affected individuals who did not carry it (P=.033). The mutation was not detected in either 566 unaffected Ashkenazi Jews from Israel or 1,022 control individuals from New York. In hospital-based series, the 1906C allele was identified in 5/463 Ashkenazi Jews with colorectal cancer, in 2/197 with endometrial cancer, and in 0/83 with ovarian cancer. When families identified by family history and in case series are included, 25 apparently unrelated Ashkenazi Jewish families have been found to harbor this mutation. Although this pathogenic mutation is not frequent in the Ashkenazi Jewish population (accounting for 2%-3% of colorectal cancer in those whose age at diagnosis is <60 years), it is highly penetrant and accounts for approximately one-third of HNPCC in Ashkenazi Jewish families that fulfill the Amsterdam criteria.     

Novel insertion 496_497insG creating a stop codon D194X in a Chinese family with X-Linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (XALD, MIM 300100), the commonest inherited peroxisomal disorder, is characterized by central nervous system demyelination, primary adrenal failure and the systemic accumulation of saturated very long chain fatty acids (VLCFAs). The defective gene ABCD1 encodes an ATP-binding cassette (ABC) transport protein named ALDP, which functions as a crucial transporter of VLCFAs into the peroxisomes for beta-oxidation. Here, we report a Chinese man with adrenomyeloneuropathy characterized by Addison's disease and spastic paraparesis. His plasma VLCFA levels, ratios of C24:0/C22:0 and C26:0/C22:0 were all significantly elevated. We performed mutation analysis of the ABCD1 gene in the proband and the family members using direct DNA sequencing and restriction analysis. A novel insertion 496_497insG in exon 1 causing a frame shift and a premature stop codon at amino acid position 194 (D194X) was identified (GenBank accession No. NM_000033). The insertional mutation abolishes an HhaI restriction site. The same mutation was found in his mother and the eldest sister even though their clinical and biochemical abnormalities were milder. Diagnosis of XALD often relies upon the detection of elevated VLCFA levels and ratios of C26:0/C22:0 and C24:0/C22:0 in fasting blood, however, 5-15% of the obligate heterozygotes would give normal values. DNA-based testing thus remains the most reliable tool for heterozygote detection when the disease-causing mutations are known. Using restriction fragment length polymorphism with HhaI, we have devised a rapid method for the identification of the carriers among the proband's family members and possibly for the screening of the mutations in other XALD patients.     

Nature and Recurrence of AVPR2 Mutations in X-linked Nephrogenic Diabetes Insipidus

X-linked nephrogenic diabetes insipidus (NDI) is a rare disease with defective renal and extrarenal arginine-vasopressin V₂ receptor responses due to mutations in the AVPR2 gene in Xq28. We analyzed 31 independent NDI families to determine the nature and recurrence of AVPR2 mutations. Twenty-one new putative disease-causing mutations were identified: 113delCT, 253del35, 255del9, 274insG, V88M, R106C, 402delCT, C112R, Y124X, S126F, W164S, S167L, 684delTA, 804insG, W284X, A285P, W293X, R337X, and three large deletions or gene rearrangements. Five other mutations—R113W, Y128S, R137H, R181C, and R202C—that previously had been reported in other families were detected. There was evidence for recurrent mutation for four mutations (R113W, R137H, S167L, and R337X). Eight de novo mutation events were detected (274insG, R106C, Y128S, 167L [twice], R202C, 684delTA, and R337X). The origins were maternal (one), grandmaternal (one), and grandpaternal (six). In the 31 NDI families and 6 families previously reported by us, there is evidence both for mutation hot spots for nucleotide substitutions and for small deletions and insertions. More than half (58%) of the nucleotide substitutions in 26 families could be a consequence of 5-methylcytosine deamination at a CpG dinucleotide. Most of the small deletions and insertions could be attributed to slipped mispairing during DNA replication.     

Krit1 missense mutations lead to splicing errors in cerebral cavernous malformation

At least 40% of families affected with cerebral cavernous malformation have a mutation in Krit1. We previously identified two point mutations in Krit1 leading to changes in amino acids (D137G and Q210E) in two different families. Further RNA analysis reveals that both point mutations actually activate cryptic splice-donor sites, causing aberrant splicing and leading to a frameshift and protein truncation. To date, no simple missense mutations have been detected in Krit1.     

Novel missense MTTP gene mutations causing abetalipoproteinemia

Objective

The microsomal triglyceride transfer protein (MTTP) plays a critical role in the formation of hepatic very low density lipoprotein. Abetalipoproteinemia (ABL) is a rare, naturally occurring extreme form of MTTP inhibition, which is characterized by the virtual absence of apolipoprotein (apo) B-containing lipoproteins in blood. The goal of this study was to examine the effect that four novel MTTP missense mutations had on protein interactions, expression and lipid-transfer activity, and to determine which mutations were responsible for the ABL phenotype observed in two patients.

Approach and results

In two patients with ABL, we identified in MTTP a novel frameshift mutation (K35Ffs*37), and four novel missense mutations, namely, G264R, Y528H, R540C, and N649S. When transiently expressed in COS-7 cells, all missense MTTP mutations interacted with apoB17, apoB48, and protein disulfide isomerase. Mutations Y528H and R540C, however, displayed negligible levels of MTTP activity and N649S displayed a partial reduction relative to the wild-type MTTP. In contrast, G264R retained full lipid-transfer activity.

Conclusions

These studies indicate that missense mutations Y528H, R540C, and N649S appear to cause ABL by reducing MTTP activity rather than by reducing binding of MTTP with protein disulfide isomerase or apoB. The region of MTTP containing amino acids 528 and 540 constitutes a critical domain for its lipid-transfer activity.     

Characterization of a germline mosaicism in families with Lowe syndrome, and identification of seven novel mutations in the OCRL1 gene.

The oculocerebrorenal syndrome of Lowe (OCRL) is an X-linked disorder characterized by major abnormalities of eyes, nervous system, and kidneys. Mutations in the OCRL1 gene have been associated with the disease. OCRL1 encodes a phosphatidylinositol 4, 5-biphosphate (PtdIns[4,5]P2) 5-phosphatase. We have examined the OCRL1 gene in eight unrelated patients with OCRL and have found seven new mutations and one recurrent in-frame deletion. Among the new mutations, two nonsense mutations (R317X and E558X) and three other frameshift mutations caused premature termination of the protein. A missense mutation, R483G, was located in the highly conserved PtdIns(4,5)P2 5-phosphatase domain. Finally, one frameshift mutation, 2799delC, modifies the C-terminal part of OCRL1, with an extension of six amino acids. Altogether, 70% of missense mutations are located in exon 15, and 52% of all mutations cluster in exons 11-15. We also identified two new microsatellite markers for the OCRL1 locus, and we detected a germline mosaicism in one family. This observation has direct implications for genetic counseling of Lowe syndrome families.