Identification of recurrent and novel mutations in the LDL receptor gene in Japanese familial hypercholesterolemia

We used the denaturing gradient gel electrophoresis (DGGE) method to investigate 120 Japanese patients with familial hypercholesterolemia (FH) for mutations in the promoter region and the 18 exons and their flanking intron sequence of the low density lipoprotein (LDL) receptor gene. Fourteen aberrant DGGE patterns were found, and the underlying mutations were characterized by DNA sequencing. Five novel missense mutations (C317S, F382L A410T, L547V, and E693K), two nonsense mutations (W512X and K790X), four frameshift mutation (355del7, 1246ins5, 1687ins1, and 2035ins1), one splicing mutation (1845+2 T→C), and two inframe mutations (661ins21 and 1115del9/ins6) were identified. Six of these mutations (L547V, E693K, W512X, 355del7, 1687ins1, and 20354ins1) have not been described before in FH. These newly identified mutations cosegregated in their family members with defective LDL receptor activity and hypercholesterolemia, and are thought to be causal for the FH phenotype. These results demonstrate that there is a broad spectrum of mutations in the LDL receptor gene in the Japanese population. Hum Mutat 14:87, 1999. © 1999 Wiley‐Liss, Inc.     

Three novel small deletion mutations of the LDL receptor gene in Korean patients with familial hypercholesterolemia.

The low-density lipoprotein (LDL) receptor gene from 80 unrelated Korean patients with familial hypercholesterolemia (FH) was analyzed to screen for small structural rearrangements that could not be detected by Southern blot hybridization. Three different small deletions were detected in exon 11 of 3 FH patients and were characterized by DNA sequence analysis. Of them two mutations are in-frame 36-bp (FH 2) and 9-bp (FH 34) deletions that result in the loss of twelve amino acids (from Met510 to Ile521) and three amino acids (Thr513, Asp514 and Trp515), respectively. Both mutations are located in the third of the five YWTD motifs of the LDL receptor gene. The third mutation (FH 400) is a 2-bp deletion that shifts the translational reading frame and results in a prematurely terminated receptor protein. The generation of a 36-bp deletion can be explained by the formation of a hairpin-loop structure mediated by inverted repeat sequences. On the other hand, the mechanism responsible for the 9- and the 2-bp deletions is probably strand-slippage mispairing mediated by short direct repeats. All of these three deletions are novel mutations. Each of the three deletions was detected only in a single pedigree out of 80 FH families analyzed.     

Two novel mutations consisting in minor gene rearrangements in the human low density lipoprotein receptor gene in Italian patients affected by familial hypercholesterolemia. Mutations in brief no. 194. Online

Mutations in the low density lipoprotein (LDL)-receptor gene cause familial hypercholesterolemia (FH), an autosomal dominant disease associated to an increased risk of premature atherosclerosis. We describe two novel mutations found in Italian families and consisting in minor gene rearrangements. The first one (FH-Pisa) is a tetranucleotide insertion occurring in exon 8, which causes a frameshift and a premature stop codon. The second one (FH-Chieti3) occurs at the 3'-end of exon 4 and consists in a trinucleotide deletion replaced by a six-base insertion, so that the reading frame is maintained with a glutamic acid-to-cysteine substitution at codon 207 and the insertion of a lysine at codon 208. Both mutations occur in regions of the LDL-receptor gene which can be considered hotspots for minor rearrangements.     

Two novel missense mutations in the LDL receptor gene causing familial hypercholesterolemia

We have employed analysis of single-strand conformation polymorphisms to identify mutations in the low density lipoprotein receptor gene causing familial hypercholesterolemia. Two familial hypercholesterolemia heterozygotes had abnormal single-strand conformation polymorphism patterns of exons 4 and 8. DNA sequencing revealed that the abnormal pattern of exon 4 was due to heterozygosity (G/T) at nucleotide 502. Nucleotide 502 is the first base of codon 147, and the G→T mutation (D147Y) changes this codon from Asp_GAC to Tyr_UAC. The abnormal pattern of exon 8 was due to heterozygosity (A/G) at nucleotide 1097. Nucleotide 1097 is the second base of codon 345, and the A→G mutation (Q345R) changes this codon from Gln_CAG to Arg_CGG- Based upon screening of 437 unrelated familial hypercholesterolemia heterozygotes, both D147Y and Q345R account for about 0.5% of the mutations causing familial hypercholesterolemia in Norway.     

Two novel mutations in the LDL receptor gene: common causes of familial hypercholesterolemia in a Spanish population

In our investigation of the LDL receptor gene in 30 Spanish patients, who were clinically diagnosed as heterozygous FH and were unrelated, we have applied single strand conformation polymorphism (SSCP) analysis and solid-phase sequencing. We identified two novel pathogenic mutations accounting for one third of the FH in this patient sample. Six patients were found to have a G to T substitution at nucleotide position 91 in exon 2 that results in a stop codon, E10X. Four patients were found to have a G deletion at nucleotide 518 in exon 4, causing a translational frameshift and a stop codon, 518delG. We have developed two polymerase chain reaction (PCR) based assays to detect easily these two mutations in all the available family members. We found fourteen E10X mutation carriers and eighteen 518delG mutation carriers. There was no statistically significant difference in mean lipid levels between carriers of these two mutations. Furthermore, haplotype analysis revealed that all E10X mutation carriers had the allele determined by TaqI-, Stul+, Avall+, Ncol- and all 518delG mutation carriers had the haplotype TaqI-, Stul+, Avall-, NcoI+. This indicates that both mutations may have been inherited from common ancestors, respectively.     

Mutation analysis in 36 unrelated Spanish subjects with familial hypercholesterolemia: identification of 3 novel mutations in the LDL receptor gene

We used the single strand conformation polymorphism (SSCP) method to investigate 36 apparently unrelated Spanish patients with familial hypercholesterolemia (FH) for mutations in the promoter region and the 18 exons and their flanking intron sequences of the low density lipoprotein receptor (LDLR) gene. Nineteen aberrant SSCP patterns were found, and the underlying mutations were characterized by DNA sequencing. In addition, we tested all patients for the presence of mutations in the gene coding for apolipoprotein B (apo B). Five missense mutations (Q71E, S156L, E256K, N543H and T705I), four nonsense mutations (W(-18)X, E10X, Q133X and C255X), six frameshift mutations (211delG, 518delG, 1045delC, 2085del19, 2207insT and 2393del9) and five splicing mutations (313+1G->C, 1061-8T->C, 1845+1G->C, 2140+5G->A and 2390-1G->C) were identified in the LDLR gene. In total, we detected 20 mutations, 3 of which, designated 1045delC, 1845+1G->C and 2207insT, have not been previously described. Seven patients were found to carry two different mutations in the same allele: W(-18)X and E256K (one patient), Q71E and 313+1G->C (two patients), 1061-8T->C and T705I (two patients), 518delG and 2140+5G->A (one patient) and N543H and 2393del9 (one patient). As we expected, there is a broad spectrum of mutations in the LDLR gene, given the genetic heterogeneity of the Spanish population.     

Twelve novel RB1 gene mutations in patients with hereditary retinoblastoma. Mutations in brief no. 206. Online

Hereditary predisposition to retinoblastoma is caused by germline mutations in the RB1 gene. Mutation analysis in this gene is important because knowledge of the causative mutation is often required for accurate risk prediction in relatives. We have performed RB1 gene mutation analysis in 45 patients with hereditary retinoblastoma. Screening by heteroduplex and SSCP analysis resulted in the identification of small mutations in 28 (62%) patients. Recurrent mutations, mostly CpG-transitions, were found in 16 patients. Two patients with isolated bilateral retinoblastoma showed missense mutations, S567L and C712R, which have previously been reported in a patient with bilateral tumors and in a family with low penetrance, respectively. Twelve of the mutations identified here have not been reported to date. These include a novel missense mutation, L662P, which was identified in two bilaterally affected siblings and their mother with unilateral retinoma.     

Identification of four novel mutations of the low-density lipoprotein receptor gene in Korean patients with familial hypercholesterolemia

To obtain insight into the genetic variation of the low-density lipoprotein (LDL) receptor gene in Korean patients with familial hypercholesterolemia (FH), we used single-strand conformation polymorphism to screen all 18 exons and a promotor of the LDL receptor gene in 20 unrelated Korean FH patients. Four novel point mutations were detected in 5 FH patients and were characterized by sequence analysis. Of them, one is a nonsense mutation, a Glu-->Stop (CAG-->TAG) at codon 161, and results in a large deletion. The other three, which were a Ala-->Glu (GCG-->GAG) mutation at signal peptide, Cys-->Tyr (TGC-->TAC) at codon 210, and Pro-->Leu (CTG-->CCG) at codon 584, were novel missense mutations, which modified the highly conserved region of the LDL receptor gene. All these mutations were absent in normolipidemic controls and were associated in heterozygote carriers with clinical signs of FH. Identification of these novel mutations provides another example of the molecular heterogeneity of the LDL receptor gene mutations causing FH.     

Mutation analysis in 46 German families with familial hypercholesterolemia: identification of 8 new mutations. Mutations in brief no. 226. Online

In order to obtain a survey of the mutations being prevalent in Northern Germany and to enable molecular genetic testing for families with clinically diagnosed familial hypercholesterolemia (FH), we screened 46 unrelated German individuals with elevated LDL levels for mutations in the 18 exons and their flanking intron sequences including the promotor region of the LDL receptor (LDLR) gene. In addition, we tested all patients for the presence of mutations in the gene coding for apolipoprotein B-100 (apoB-100). We detected 15 mutations affecting the LDLR gene, 8 of which, designated A29S, 195insAT, 313+1insG, 553insG, 680insGGACAAATCTG, D200N, E267K and L411V have not yet been reported. One patient is heterozygous for the double mutant N543H and 2393del9Bp. Two patients carried the mutation R3500Q (Arg-->Glu) within the apoB-100 gene.     

Identification of novel PAX6 mutations in two families with bilateral aniridia. Mutations in brief no. 167. Online

We report two novel PAX6 mutations in aniridia patients of two Swiss pedigrees (We, Sc) which give rise to different phenotypes. An SSCP analysis of the PAX6 14 exons reveals electrophoretic mobility shifts exclusively in exons 5 and 12 of aniridia patients. As determined by bidirectional sequencing and restriction digest analysis, these shifts are caused by mono-allelic base transitions in exon 5 (c.547C-->T; R44X; We) and intron 12 (IVS12+5G-->A; Sc). Each mutation co-segregates with the trait in the affected family with complete penetrance. The Sc mutation in the splicing donor site of intron 12 may result in either intron inclusion or exon skipping, both giving rise to a truncated PAX6 protein which may retain a residual transactivating activity. In contrast, the We genetic alteration is a loss-of-function mutation leading to a more severe phenotype than that observed in the Sc pedigree.     

Molecular genetics of the LDL receptor gene in familial hypercholesterolemia.

The low density lipoprotein (LDL) receptor is a cell surface transmembrane protein that mediates the uptake and lysosomal degradation of plasma LDL, thereby providing cholesterol to cells. Mutations disrupting the function of this receptor produce autosomal dominant familial hypercholesterolemia (FH). Affected individuals have elevated plasma levels of LDL, which causes premature coronary atherosclerosis. To date, 71 mutations in the LDL receptor gene have been characterized at a molecular level. In this report, we describe 79 additional mutations and review the insights that all 150 mutations have provided into the structure/function relationship of the receptor protein and the clinical manifestations of FH.     

Identification of 9 novel IDS gene mutations in 19 unrelated Hunter syndrome (mucopolysaccharidosis Type II) patients. Mutations in brief no. 202. Online

Hunter syndrome is an X-linked lysosomal storage disorder caused by a deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS). The IDS deficiency can be caused by several different types of mutations in the IDS gene. We have performed a molecular and mutation analysis of a total 19 unrelated MPS II patients of different ethnic origin and identified 19 different IDS mutations, 9 of which were novel and unique. SSCP analysis followed by DNA sequencing revealed four novel missense mutations: S143F, associated with the 562C-->T polymorphism, C184W, D269V and Y348H. Two novel nonsense mutations were found: Y103X (433C-->A) and Y234X (826C-->G). In two patients two novel minor insertions (42linsA and 499insA) were identified. In one patient a complete IDS deletion was found, extending from locus DXS1185 to locus DXS466).     

Two novel mutations of the arylsulfatase B gene in two Italian patients with severe form of mucopolysaccharidosis. Mutations in brief no. 127. Online

Mucopolysaccharidosis type VI (MPS VI) or Maroteaux-Lamy syndrome, is a autosomal recessive disorder, due to the deficiency of the lysosomal enzyme N-acetylgalactosamine-4-sulfatase (arylsufatase B, ASB: EC 3.1.6.12). Three classical forms of the disease have been differentiated: severe, intermediate, mild. Mutational analysis of the ASB gene resulted in the identification of 30 ASB mutant alleles, each of which was found to be unique among unrelated patients, demonstrating a broad molecular heterogeneity of the disease. In this communication we present two novel mutant alleles in two severely affected subjects. Both alterations, the missense mutation G302R and the nonsense Q456X, were found in homozygosity and were confirmed by amplification refractory mutation system (ARMS) or restriction analysis. The missense G302R mutation concerns an amino acid which may be of special importance to the polypeptide, since 302 position is completely conserved in all the eukaryotic sulfatases aligned so far; the nonsense mutation Q456X leads to the translation of a putative mutant ASB protein lacking the last 78 amino acids with a loss of the 8 kD mature polypeptide, one of the two peptides generated by intralysosomal proteolytic processing of the 64kD precursor.     

Identification of three novel mutations in the dystrophin gene detected by the heteroduplex/SSCA screening procedure. Mutations in brief no. 222. Online

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked neuromuscular disorders associated with alterations in the dystrophin gene. Analysis of 45 DMD/BMD patients has identified 18 patients with no deletion in the dystrophin gene. Heteroduplex analysis (HD), single strand conformation analysis (SSCA), and subsequent sequencing, identified five mutations and nine polymorphisms. Three out of the 5 mutations (780C>G, 2501-1g-->t, 9812 9813ins9800-9812) are first reported here. Furthermore we compare the relative efficiencies of the two alternatives methods (HD and SSCA) for screening sequence alterations.     

Five familial hypercholesterolemic kindreds in Japan with novel mutations of the LDL receptor gene

In the course of investigations of familial coronary artery disease in Hokkaido, the northern island of Japan, we identified five families in which multiple members showed elevated plasma levels of low-density lipoprotein (LDL) cholesterol. To determine the genetic etiology of their lipoprotein abnormalities, we screened DNA samples from these families for mutations in all 18 exons and the exon-intron boundaries of the LDL receptor (LDLR) gene. Novel point mutations were identified in each family: (1) a C-to-A transversion at nucleotide 285, causing a nonsense mutation at codon 74, in eight members of family A; (2) a G-to-A transition at nucleotide 1136, causing substitution of Tyr for Cys at codon 358, in six members of family B; (3) a C-to-T transition at nucleotide 1822, causing substitution of Ser for Pro at codon 587, in five members of family C; (4) a one-base insertion of G to a five-G stretch at nucleotides 1774–1778 (codons 571–572), causing a frameshift, in six members of family D; and (5) a one-base deletion of T at nucleotide 1963–1964 (codon 634), causing a frameshift, in three members of family E. Through the molecular genetic approach a total of 28 individuals in these families were diagnosed unequivocally as heterozygous for the respective LDLR mutations. This method also helped us to diagnose familial hypercholesterolemia, or to exclude from carrier status, 11 children with borderline high cholesterol levels. Received: May 20, 1998 / Accepted: June 30, 1998