Mutation analysis and description of sixteen RSH/Smith-Lemli-Opitz syndrome patients: polymerase chain reaction-based assays to simplify genotyping

We report the clinical and molecular data of 16 patients with RSH/Smith-Lemli-Opitz syndrome (RSH/SLOS) with varying phenotypic severity, for which we have identified mutations in both alleles. RSH/SLOS is an autosomal recessive malformation syndrome caused by mutations in the gene encoding the sterol Delta(7)-reductase. This protein catalyzes the reduction of 7-dehydrocholesterol to cholesterol in the last step of cholesterol biosynthesis via the Kandutsch-Russell pathway. In addition to previously reported mutations (T93M, L109P, G147D, W151X, T154M, R242C, A247V, T289I, IVS8-1G-->C, Y408H, and E448K), we have identified six previously undescribed mutations (321G-->C, W177R, R242H, Y318N, L341P, and C444Y). We also report rapid polymerase chain reaction (PCR)-based assays developed to detect four of the recurring mutations (T93M, W151X, V326L, and R404C) and six other RSH/SLOS mutations (321G-->C, L109P, T154M, T289I, Y318N, and L341P). The purpose of this article is to correlate detailed clinical information with molecular data in order to improve our understanding of the genotype-phenotype correlation of RSH/SLOS and to report the development of PCR-based assays that will allow more rapid mutation analysis.     

DHCR7 genotypes of cousins with Smith‐Lemli‐Opitz syndrome

Smith‐Lemli‐Opitz syndrome (SLOS) is an autosomal recessive disorder of cholesterol biosynthesis caused by mutations of the 7‐dehydrocholesterol reductase gene (DHCR7). We report on three cousins with SLOS, all of whom were found to be compound heterozygotes for the common splice site mutation IVS8‐1G→C and the missense mutation T289I. DNA analysis of one set of parents demonstrated that the father carried the missense mutation and the mother carried the IVS8–1G→C mutation. By extension, the two unrelated mothers were both heterozygous for IVS8‐1G→C. This finding supports the notion of a high carrier frequency of the IVS8‐1G→C null mutation in Northern European Caucasians. © 2001 Wiley‐Liss, Inc.     

Detection of a common mutation in the RSH or Smith‐Lemli‐Opitz syndrome by a PCR‐RFLP assay: IVS8‐1G → C is found in over sixty percent of US propositi

The RSH or Smith‐Lemli‐Opitz syndrome (SLOS) is a relatively common autosomal recessive disorder of cholesterol biosynthesis resulting from a deficiency of the enzyme 7‐dehydrocholesterol Δ7‐reductase (7‐DHCR). Mutations in 7‐DHCR gene cause SLOS. Among these, a G → C transversion in the splice acceptor site of exon 9 (IVS8‐1G → C) was suspected to be a frequent mutation, having been detected in about 18% of SLOS patients so far. This mutation results in the elimination of a AIwN1 restriction endonuclease site. We report a simple PCR‐RFLP assay to detect the IVS8‐1 G → C mutation. Using this method, we identified the IVS8‐1G → C mutation in 21 of 33 SLOS propositi. This mutation was detected in one of 90 normal adult Caucasian Americans; but not among 121 Africans from Sierra Leone, 120 Caucasians from Finland, 95 Chinese or 103 Japanese adults. The results of this study provide further evidence that IVS8‐1G → C transversion is a very common mutation in SLOS patients from the US and that the carrier rate in US caucasians may be high. The simple PCR‐RFLP assay developed makes identification of this mutation convenient for diagnosis and for carrier detection. Am. J. Med. Genet. 90:347–350, 2000 © 2000 Wiley‐Liss, Inc.     

Smith‐Lemli‐Opitz (RHS) syndrome: holoprosencephaly and homozygous IVS8‐1G→C genotype

Smith‐Lemli‐Opitz syndrome (RHS) (SLOS, OMIM 270400) is an autosomal recessive disorder of cholesterol biosynthesis caused by mutations of the 3β‐hydroxysterol Δ⁷‐Δ⁸‐reductase gene, DHCR7. We report a fetus with holoprosencephaly and multiple congenital anomalies who was homozygous for the IVS8‐1G→C mutation. Following termination of pregnancy, both the elevated amniotic fluid 7‐dehydrocholesterol level and the DHCR7 mutations were demonstrated. Two other newborn infants with IVS8‐1G→C/IVS8‐1G→C genotype are described. This report illustrates a severe phenotypic extreme of SLOS associated with a null genotype, underscores the complex relationship between SLOS and holoprosencephaly, and discusses the possible pathogenetic mechanisms of the development of holoprosencephaly in SLOS. © 2001 Wiley‐Liss, Inc.     

Frequency gradients of DHCR7 mutations in patients with Smith-Lemli-Opitz syndrome in Europe: evidence for different origins of common mutations

Smith-Lemli-Opitz syndrome/RSH (SLOS) is a multiple congenital anomaly syndrome caused by mutations in the gene for Delta7-sterol reductase (DHCR7) which catalyses the last step in the biosynthesis of cholesterol. SLOS is among the common recessive disorders in Europeans but almost absent in most other populations. More than 40 mutations in the DHCR7 gene some of which are frequent have been described in SLOS patients of various origins. Here we report mutation analysis of the DHCR7 gene in SLOS patients from Poland (n = 15), Germany/Austria (n = 22) and Great Britain (n = 22). Altogether 35 different mutations were identified and the two null mutations IVS8-1G > C and W151X were the most frequent in the total sample. In all three populations three mutations accounted for >0.5 of SLOS chromosomes. The mutational spectra were, however, significantly different across these populations with each of the common mutations showing an east-west gradient (W151X, V326L) or vice versa (IVS8-1G > C). W151X is the most frequent (0.33) mutation in Polish SLOS patients. It has an intermediate frequency in German/Austrian patients (0.18) and is rare among British patients (0.02). V326L shows the same distribution pattern (Poland 0.23, Germany/Austria 0.18, Britain 0.02). In contrast IVS8-1G > C is most frequent in Britain (0.34) intermediate in Germany/Austria (0.20) and rare in Poland (0.03). All analysed IVS8-1G > C and V326L alleles shared the same DHCR7 haplotype, whereas the W151X mutation occurred on different haplotypes. There is evidence for both recurrent mutations and founder effects. Together this suggests that the common SLOS mutations in Europe have different geographic and historic origins and spread across the continent in opposite directions.     

Fabry disease: 20 novel GLA mutations in 35 families

Thirty two mutations have been found in 35 unrelated patients of European origin with Fabry disease, including 8 females. Twenty of the mutations are novel and comprise of 13 missense: H46Y, W47G, R49P, C94S, F113S, G258R, P259R, Q279H, Q280H, R363H, A377D, P409A, P409T; 1 nonsense: L294X; 5 small deletions: 154delT, 520delT, 909‐918del10, 1152‐1153delCA, 1235‐1236delCT and 1 splice site mutation: IVS5+2t→c. The remaining 12 mutations have all been reported previously. All patients with deletions had the classic form of the disease but it was not possible to predict the phenotype from the missense mutations. © Wiley‐Liss, Inc.     

Mutational analysis of mucopolysaccharidosis type VI patients undergoing a trial of enzyme replacement therapy

Mucopolysaccharidosis type VI (MPS VI), or Maroteaux-Lamy syndrome, is a lysosomal storage disorder caused by a deficiency of N-acetylgalactosamine-4-sulfatase (ARSB). Seven MPS VI patients were chosen for the initial clinical trial of enzyme replacement therapy. Direct sequencing of genomic DNA from these patients was used to identify ARSB mutations. Each individual exon of the ARSB gene was amplified by PCR and subsequently sequenced. Nine substitutions (c.289C>T [p.Q97X], c.629A>G [p.Y210C], c.707T>C [p.L236P], c.936G>T [p.W312C], c.944G>A [p.R315Q], c.962T>C [p.L321P], c.979C>T [p.R327X], c.1151G>A [p.S384N], and c.1450A>G [p.R484G]), two deletions (c.356_358delTAC [p.Y86del] and c.427delG), and one intronic mutation (c.1336+2T>G) were identified. A total of 7 out of the 12 mutations identified were novel (p.Y86del, p.Q97X, p.W312C, p.R327X, c.427delG, p.R484G, and c.1336+2T>G). Two of these novel mutations (p.Y86del and p.W312C) were expressed in Chinese hamster ovary cells and analyzed for residual ARSB activity and mutant ARSB protein. The two common polymorphisms c.1072G>A [p.V358M] and c.1126G>A [p.V376M] were identified among the patients, along with the silent mutation c.1191A>G. Cultured fibroblast ARSB mutant protein and residual activity were determined for each patient, and, together with genotype information, were used to predict the expected clinical severity of each MPS VI patient.     

Frequency and ethnic distribution of the common DHCR7 mutation in Smith‐Lemli‐Opitz syndrome

Smith‐Lemli‐Opitz syndrome (SLOS) is an inherited multiple malformation syndrome caused by enzymatic deficiency of 3β‐hydroxysterol‐Δ⁷‐reductase (DHCR7). SLOS is thought to be most common among European Caucasians, with an incidence of 1 in 20,000 to 1 in 30,000 births. To define the carrier rate and ethnic distribution of SLOS, we screened DNA samples from 2,978 unrelated individuals for the most common SLOS mutation (IVS8‐1G→C). Twenty‐four heterozygotes of the IVS8‐1G→C mutation were detected in 2,978 individuals of European Caucasian and Black backgrounds. For European Caucasians, the carrier rate for SLOS may be as high as 1 in 30, suggesting an incidence of 1 in 1,700 to 1 in 13,400. This high number is supported by the recent observation of newborn and prenatal incidence of 1 in 22,000 in the Caucasian population. Ours is the first report of the IVS8‐1G→C mutation in persons of African ancestry. Published 2001 Wiley‐Liss, Inc.     

Spectrum of Delta(7)-dehydrocholesterol reductase mutations in patients with the Smith-Lemli-Opitz (RSH) syndrome

The Smith-Lemli-Opitz syndrome (SLOS; also known as the RSH syndrome) is an autosomal recessive genetic disorder, leading to characteristic multi-organ developmental abnormalities, dysmorphic facies, limb malformations and mental retardation. Mutations in the gene for Delta(7)-dehydrocholesterol reductase (Delta(7)-reductase), which catalyzes the last step in cholesterol biosynthesis, cause the disease. We screened 32 patients with SLOS, 28 from the USA and four from Sweden. Twenty-two different nucleotide changes, predicted to be disease-causing mutations, were identified; 20 missense mutations, one nonsense mutation and one splice-site mutation involving the exon 9 acceptor site (IVS8 -1G-->C) were detected. All probands were heterozygous for mutations. Twelve of these mutations have not been reported previously, including missense mutations L148R, F168I, D175H, P179L, P243R, F284L, N287K, F302L, R404S, Y462H, R469P and one nonsense mutation W37X [corrected]. Coupled with previously reported mutations, these findings bring the total of different Delta(7)-reductase mutations to 36. These are distributed throughout the coding sequence of the Delta(7)-reductase gene except exons 3 and 5, with a clustering in exon 9. Three mutations account for 54% of those observed in our cohort, the splice acceptor site mutation IVS8 -1G-->C (22/64 alleles, 34%), T93M (8/64, 12.5%) and V326L (5/64, 7.8%). Severity of SLOS was negatively correlated with both plasma cholesterol and relative plasma cholesterol, but not with 7-dehydrocholesterol, the immediate precursor, confirming previous observations. However, no correlation was observed between mutations and phenotype, suggesting that the degree of severity may be affected by other factors. We estimate that between 33 and 42% of the variation in the SLOS severity score is accounted for by variation in plasma cholesterol. Thus, factors other than plasma cholesterol are additionally involved in determining severity.     

Four novel mutations in the cystathionine β‐synthase gene: Effect of a second linked mutation on the severity of the homocystinuric phenotype

Homocystinuria due to cystathionine β‐synthase (CBS) deficiency is frequently caused by missense mutations. In this article, we report four novel missense mutations in the CBS gene: 172C→T (R58W) linked in cis with A114V; 376A→G (M126V); 904G→A (E302K); and 1006C→T (R336C). The CBS activity of the corresponding mutant enzymes expressed in Escherichia coli was greatly diminished, confirming the pathogenicity of these mutations. Western analysis showed that the R58W+A114V and M126V mutant enzymes were unstable in E. coli, while the E302K subunits were partially degraded to shorter products. Using site‐directed mutagenesis we found that CBS containing either the R58W or A114V as the only mutations demonstrated 18% and 46% of normal activity, respectively. Both mutant forms of CBS were stable in E. coli. When these two mutations were expressed in cis, the resultant mutant protein exhibited activity 1.3% that of a control. All these in vitro results were in good agreement with the clinical manifestation in these patients. The Italian patient 2241, an A114V+R58W/M126V compound heterozygote, exhibited severe pyridoxine nonresponsive homocystinuria, while another Italian patient 2242, with an A114V/E302K genotype, responded to pyridoxine treatment and had a much milder phenotype. The third patient 3064, an English compound heterozygote for two severe mutations R336C and G307S, was B6 nonresponsive. This report of a ninth homocystinuric allele carrying two mutations in cis raises the possibility that double mutant alleles may be underestimated in homocystinuric patients. In this context, a search for additional mutations in cis may sometimes be necessary to establish a good genotype‐phenotype relationship. Hum Mutat 13:453–457, 1999. © 1999 Wiley‐Liss, Inc.     

Identification of 12 novel mutations and two new polymorphisms in the arylsulfatase A gene: Haplotype and genotype–phenotype correlation studies in spanish metachromatic leukodystrophy patients

Arylsulfatase A (ARSA) deficiency is the main cause of metachromatic leukodystrophy (MLD), a lysosomal disorder with no specific treatment. In view of the importance of genetic counseling, analyses of mutations and polymorphisms, including the ARSA pseudodeficiency allele, were carried out in 18 unrelated Spanish MLD patients. A systematic search allowed us to identify 100% of the alleles involving 17 different mutations, 12 of which are novel: G32S, L68P, R84W, P94A, G99V, P136S, W193X, H227Y, R288H, G308D, T327I, and IVS6‐12C→G. Two new polymorphisms, 2033C>T and 2059C>T, were identified in intron 6 which, in combination with two polymorphisms previously described (2161C>G and 2213C>G), gave rise to four different haplotypes in the control population. In addition, we also studied polymorphism 842G>T. Linkage disequilibrium was detected between mutations IVS2+1G→A, D255H, and T327I and specific haplotypes, suggesting a unique origin for these mutations. Moreover, mutation T327I was always associated with the T allele of the new rare variant A210A (893C>T). The distribution of mutation D255H (frequency 19.4%) among patients with different MLD clinical presentation revealed a clear genotype–phenotype correlation paralleling that reported for mutation IVS2+1G→A (frequency 25%). Among the novel mutations, only P136S and R288H occurred on a background of the ARSA pseudodeficiency allele. Screening 182 normal chromosomes identified a frequency of 8.8% of this allele; moreover, we identified two unrelated subjects with the polyA‐ mutation in the absence of the N350S mutation, and this infrequent haplotype reinforced the heterogeneity of conditions with ARSA deficiency. Hum Mutat 14:240–248, 1999. © 1999 Wiley‐Liss, Inc.     

Metachromatic leukodystrophy: Identification of the first deletion in exon 1 and nine novel point mutations in the arylsulfatase A gene

Metachromatic leukodystrophy (MLD), a lysosomal storage disease caused by the deficiency of arylsulfatase A (ASA), is inherited as an autosomal recessive trait, and its frequency is estimated to be 1 in 40,000 live births. Genomic DNA from 21 MLD patients (14 late-infantile and 7 juvenile cases) was amplified in four overlapping PCR fragments and tested by allele-specific oligonucleotide (ASO) for the two common mutations 459+1G→A and P426L. These mutations were found in only 28.6% of the alleles studied. The remaining alleles were analyzed by chemical mismatch cleavage (CMC) and automatic sequencing. In addition to five previously reported mutations (459+1G→a, A212V, R244C, R390W, P426L), 10 novel mutations were identified: 9 missense mutations (S95N, G119R, D152Y, R244H, S250Y, A314T, R384C, R496H, K367N) and one 8 bp deletion in exon 1, the first mutation reported in this exon. These methods allowed us to identify 76% of the alleles tested. Genotype-phenotype correlations could be established for some of these mutations. These results confirm the heterogeneity of mutations causing MLD and suggest that CMC is a reliable and informative screening method for point mutation detection in the arylsulfatase A gene. Hum Mutat 9:234–242, 1997. © 1997 Wiley-Liss, Inc.     

Fabry disease: 45 novel mutations in the alpha-galactosidase A gene causing the classical phenotype

The nature of the molecular lesions in the alpha-galactosidase A (alpha-Gal A) gene causing Fabry disease was determined in 50 unrelated families with the classic phenotype of this X-linked recessive lysosomal storage disease. Genomic DNA was isolated from affected males or obligate carrier females, and the entire alpha-Gal A coding region as well as the flanking and intronic sequences were analyzed by PCR amplification and automated sequencing. Forty-five new mutations were identified including 38 single base substitutions (32 missense and four nonsense) and nine gene rearrangements: MIR, M42T, G43D, G43V, H46Y, F50C, L68F, G132R, T141I, Y152X, K168R, G183S, V199M, P205R, Y207S, Q221X, C223R, C223Y, D234Y, G271C, A288P, P293A, R301G, I303N, I317T, E341D, P362L, R363C, R363H, G373D, I384N, T385P, Q396X, E398K, S401X, P409A, g7325insC, g7384del13, g8341delG, g8391del4/ins3, g10511delTAGT, g10704delACAG, g11019insG, g11021insG, and g11048delAGG. In the remaining five Fabry families, four previously reported mutations were detected (W81X, R112C, g11011delTC, and g11050delGAG) of which the R112C substitution was found in two families who were unrelated by haplotyping. These studies further define the heterogeneity of mutations in the alpha-Gal A gene causing the classical Fabry disease phenotype, and permit precise carrier detection and prenatal diagnosis in these families.     

Novel 7‐DHCR mutation in a child with Smith‐Lemli‐Opitz syndrome

Smith‐Lemli‐Opitz syndrome (SLOS) is an autosomal recessive disorder characterized by minor facial anomalies, mental retardation, and multiple congenital abnormalities. Biochemically, the disorder is caused by deficient activity of 7‐dehydrocholesterol reductase, which catalyzes the reduction of the Δ7 double bond of 7‐dehydrocholesterol to produce cholesterol. Recently, mutations in the gene encoding 7‐dehydrocholesterol reductase (7DHCR) were found to cause SLOS. We report the first molecular characterization of an Italian SLOS patient. Interestingly, his paternal 7DHCR allele, of Arab origin, harbored a novel P329L mutation which in combination with a maternal splice‐site (IVS8‐1 G>C) mutation resulted in a relatively milder phenotype. Am. J. Med. Genet. 91:138–140, 2000. © 2000 Wiley‐Liss, Inc.     

Characterization of eleven novel mutations (M45L, R119H, 544delG, G145V, H154Y, C184Y, D289V, 862+5A, 1172delC, R411X, E459K) in the tissue-nonspecific alkaline phosphatase (TNSALP) gene in patients with severe hypophosphatasia. Mutations in brief no. 217. Online

Hypophosphatasia is a rare inherited disorder characterized by defective bone mineralization and deficiency of serum and tissue liver/ bone/kidney tissue alkaline phosphatase (L/B/K ALP) activity. We report the characterization of tissue-nonspecific alkaline phosphatase (TNSALP) gene mutations in a series of 9 families affected by severe hypophosphatasia. Fourteen distinct mutations were found, 3 of which were previously reported in the North American or Japanese populations. Seven of the 11 new mutations were missense mutations (M45L, R119H, G145V, C184Y and H154Y, D289V, E459K), the four others were 2 single nucleotide deletions (544delG and 1172delC), a mutation affecting donor splice site (862 + 5A) and a nonsense mutation (R411X).     

Homozygosity for the W151X stop mutation in the Δ7‐sterol reductase gene (DHCR7) causing a lethal form of Smith‐Lemli‐Opitz syndrome: Retrospective molecular diagnosis

Smith‐Lemli‐Opitz syndrome (SLOS) is a multiple congenital anomalies syndrome caused by an abnormality in cholesterol metabolism. The clinical severity may vary from very mild to lethality in utero, making diagnosis difficult at both ends of the spectrum. Patients with severe SLOS might often escape diagnosis because they die before the correct diagnosis is made. We describe an Austrian family whose first child died neonatally with multiple congenital anomalies. The second pregnancy was terminated because the fetus showed similar severe anomalies ultrasonographically. A further pregnancy ended in a spontaneous first trimester abortion. Clinical diagnosis of SLOS was not considered until the autopsy of the fetus of the terminated pregnancy. Because no material for biochemical testing was available we performed mutational analysis of the DHCR7 gene from paraffin‐embedded tissue and a Guthrie card focusing on mutations known to cause a severe SLOS phenotype. This demonstrated homozygosity for the mutation W151X, which has been demonstrated to be a functional null mutation. Our data confirm the concept that homozygosity for functional null alleles of the DHCR7 locus results in intrauterine or perinatal lethality. Furthermore, our findings suggest the usefulness of molecular studies of stored material in similarly affected cases where no material for biochemical analysis is available. Am. J. Med. Genet. 95:174–177, 2000. © 2000 Wiley‐Liss, Inc.     

Pharmacological rescue of carnitine transport in primary carnitine deficiency

Primary carnitine deficiency is a recessive disorder caused by heterogeneous mutations in the SLC22A5 gene encoding the OCTN2 carnitine transporter. Here we extend mutational analysis to eight new families with this disorder. To determine the mechanism by which missense mutations impaired carnitine transport, the OCTN2 transporter was tagged with the green fluorescent protein and expressed in CHO cells. Analysis by confocal microscopy indicated that several missense mutants (M1I, R169W, T232 M, G242 V, S280F, R282Q, W283R, A301D, W351R, R399Q, T440 M, E452 K, and T468R) matured normally to the plasma membrane. By contrast, other mutations (including R19P, DeltaF22, R83L, S280F, P398L, Y447C, and A142S/R488 H) caused significant retention of the mutant OCTN2 transporter in the cytoplasm. Failed maturation to the plasma membrane is a common mechanism in disorders affecting membrane transporters/ion channels, including cystic fibrosis. To correct this defect, we tested whether drugs reducing the efficiency of protein degradation in the endoplasmic reticulum (ER) (phenylbutyrate, curcumin) or capable of binding the OCTN2 carnitine transporter (verapamil, quinidine) could improve carnitine transport. Prolonged incubation with phenylbutyrate, quinidine, and verapamil partially stimulated carnitine transport, while curcumin was ineffective. These results indicate that OCTN2 mutations can affect carnitine transport by impairing maturation of transporters to the plasma membrane. Pharmacological therapy can be effective in partially restoring activity of mutant transporters.     

Novel mutations in the 7-dehydrocholesterol reductase gene of 13 patients with Smith–Lemli–Opitz syndrome

Smith–Lemli–Opitz syndrome (SLOS) is caused by mutations in the DHCR7 gene leading to deficient activity of 7-dehydrocholesterol reductase (DHCR7; EC 1.3.1.21), the final enzyme of the cholesterol biosynthetic pathway, resulting in low cholesterol and high concentrations of its direct precursor 7-dehydrocholesterol in plasma and tissues. We here report mutations identified in the DHCR7 gene of 13 children diagnosed with SLOS by clinical and biochemical criteria. We found a high frequency of the previously described IVS8–1 G > C splice acceptor site mutation (two homozygotes, eight compound heterozygotes). In addition, 13 missense mutations and one splice acceptor mutation were detected in eleven patients with a mild to moderate SLOS-phenotype. The mutations include three novel missense mutations (W182L, C183Y, F255L) and one novel splice acceptor site mutation (IVS8–1 G > T).
Two patients, homozygous for the IVS8–1 G > C mutation, presented with a severe clinical phenotype and died shortly after birth. Seven patients with a mild to moderate SLOS-phenotype disclosed compound heterozygosity of the IVS8–1 G > C mutation in combination with different novel and known missense mutations.