Bertolini S

Calandra S

Casarino L

Cassanelli S

De Stefano F

Elicio N

Naselli A

Rolleri M

Calandra S.

BERTOLINI, STEFANO

DE STEFANO, FRANCESCO

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Copyright Q Munksgaard 1998 CLINICAL GENETICS ISSN wO9-9163 Short Report A ‘de novo’ point mutation of the low-density lipoprotein receptor gene in an Italian subject with primary hypercholesterolemia Cassanelli S .  Bertolini S ,  Rolleri M, De Stefan0 F, Casarino L, Elicio N. Naselli A, Calandra S .  A ‘de nouo‘ point mutation of the low-den- sity lipoprotein receptor gene in an Italian subject with primary hyperc- holesterolemia. Clin Genet 1998: 53: 391-395. 0 Munksgaard, 1998 Severe hypercholesterolemia was found in an 1 1-year-old boy with no family history of familial hypercholesterolemia. The reduced LDL-re- ceptor activity in cultured skin fibroblasts (40% “’I-LDL degradation as compared with a control cell line) indicated the presence of an LDL-receptor defect. The analysis of the promoter region and the ex- ons of LDL-receptor gene by single strand conformation polymorphism revealed an abnormal migration pattern in exon 1, which was due to a T-*A transversion at nucleotide 28 of the cDNA. This novel mutation causes an arginine for tryptophane substitution at position - 12 of the signal peptide (W-12R) and introduces an AuiII restriction site in exon 1. Screening of the mutation by polymerase chain reaction (PCR) am- plification of exon 1 and AuiII  digestion revealed that none of the proband’s family members carried the mutation. Non-paternity was ex- cluded after the analysis of a battery of 14 short tandem repeats lo- cated in 13 different chromosomes. These results are consistent with the hypothesis that the proband is heterozygous for a ‘de nouo’ mutation of the LDL-receptor gene producing a non-conservative amino acid substitution. We suggest that the change in the net charge of the signal peptide, caused by the addition of a positively charged amino acid, impairs the co-translational translocation of the nascent receptor I protein across the endoplasmic reticulum membrane. S Cassanelli”, S Bertolinib, M Rollerib, F De Stefanoc, L Casarino’, N Eliciob, A Nasellid and S Calandra” a Department of Biomedical Science, Section of General Pathology, University of Modena, Atherosclerosis Prevention Centre, Department of Internal Medicine, Institute of Forensic Medicine, Institute ‘G. Gaslini’, University of Genoa, Italy Key words: ‘de novo’ mutation - low-density lipoprotein receptor - primary hypercholesterolemia - signal peptide Corresponding author: Sebastiano Calan- dra, Sezione di Patologia Generale, Diparti- mento di Scienze Biomediche, Universita di Modena, Via Campi 287, 41 lo0 Modena, Italy. Fax: t 39 59 428103; e-mail: seb- cal@unimo.it Received 8 January 1998, revised and accepted for publication 2 February 1998 Familial hypercholesterolemia (FH) is an autoso- ma1 co-dominant disorder due to defects of the LDL-receptor (LDL-R) function caused by muta- tions of the LDL-R gene (1). Up  to now more than 200 mutations of this gene have been reported in FH subjects (1). During the analysis of LDL-R gene mutations in Italian FH patients we have identified more than 50 different mutations (in- cluding major rearrangements, missense, nonsense, frameshift mutations and splicing defects). In all F H  families examined so far we have been able to demonstrate the vertical transmission of the LDL- R gene mutation and/or the presence of the muta- tion in proband’s first degree relatives with FH. During this survey we found a hypercholes- terolemic subject who appears to carry a ‘de n o m ’  mutation of the LDL-R gene. Materials and methods Proband The proband (AN), a 16-year-old boy, was found to have hypercholesterolemia (9 mmol/l) at the age of 11 during a routine blood test. After the exclu- sion of all secondary causes of hyperlipoproteine- mia he was suspected to have familial hypercholesterolemia as a consequence of moder- ately elevated plasma cholesterol in his father. 391 Cassanelli et al. Table 1. Clinical and biochemical features of proband’s family Subject Age CH LDL-CH HDL-CH TG Apo A1 ApoB (years) (mmoVl) (mgidl) Proband 16 9.90 8.22 0.90 1.70 112 195 Father 40 5.97 4.42 1.16 0.86 138 120 Mother 40 4.32 2.69 1.41 0.49 165 80 Brother 19 4.19 2.38 1.24 1.25 142 72 Paternal grandmother 63 9.66 5.00 1.96 3.75 - - Analysis of LDL-R gene DNA was extracted from peripheral blood (2). Southern blot and SSCP analysis of all exons and the proximal promoter of the LDL-receptor gene were performed as reported previously (3, 4). For the amplification of exon 1 the following primers were used: 5’-ACT CCT CCC CCT GCT AGA AAC CTC A-3’ (forward primer) and 5’-TTC (reverse primer). The amplification conditions were the following: 94°C for 1 min and 72°C for 1 min for 30 cycles. PCR amplified exon 1 was sequenced directly (Thermo Sequenase Kit, Amersham, UK) (4), using one of the primzrs specified above (re- verse primer). For screening of LDL-R mutation found in proband AN (TGG + AGG), exon 1 was amplified as specified above and digested with the restriction enzyme AviII. DNA fragments were separated by 2% agarose gel electrophoresis and stained with ethidium bromide. TGG CGC CTG GAG CAA GCC TTA C-3’ Fibroblast culture and LDL-receptor activity A skin biopsy was taken from the proband. Ex- plants were cultured in 25-cm2 flasks in DMEM (Dulbecco’s modification of Eagle’s medium), 100 IU/ml of penicillin, and 50 pg/ml of streptomycin, 2 mM glutamine, 15% fetal calf serum, and 95% air-5% CO,. The assay of lZ51-LDL binding, inter- nalization and degradation by cultured skin fibrob- lasts was performed as previously described ( 5 ) .  Paternity testing A small aliquot (10 ng) of DNA extrated from peripheral blood was amplified by PCR (6, 7). Three multiplex PCR reactions were performed for the following markers: HumFl3A01, HumTHO 1, HumVWA31/A, HumFES/FPS, D3S1358, D19S253, HumF13B, HumTPOX, HumCSFlPO, HumLPL, HumFGA, D6S502, D18S51, D21S11 (6, 7). An aliquot of the amplification reaction (0.5-2 p1) was mixed with 6 fmoles of commercial internal standard GS 2500 (ABD, USA), dena- 392 tured at 90°C for 2 min and loaded onto a 6% polyacrylamide denaturing gel. Samples were elec- trophoresed at constant power (36 W) for 5 h in an Applied Biosystems Automated DNA sequencer 373 A Leon and sized by Genescan 672 software (ABD, USA), employing the Local Southern method. Informed consent was obtained from all subjects. Results Table 1 shows the clinical features and plasma lipids in the proband’s family. While serum choles- terol levels of the proband’s mother and brother were within the normal range, those of the proband’s father and paternal grandmother were slightly above the mean level found in age matched controls (5.59 0.91 mmol/l). LDL receptor activ- ity ( ‘251-LDL degradation in cultured skin fibrob- lasts) in the proband’s fibroblasts was 40% of the value found in a control cell line, thus indicating that the proband had an LDL-receptor defect. Southern blot analysis of the LDL-R gene ruled out the presence of a major rearrangement, while SSCP analysis revealed the presence of an abnor- mal migration pattern in exon 1. Direct sequencing of this exon (Fig. 1) showed that the proband was heterozygous for a T +A transversion at nucle- otide 28 of the LDL-receptor cDNA, a mutation that causes an arginine for tryptophane substitu- tion at position - 12 of the signal peptide (W- 12R) of the LDL-receptor. No other mutation was discovered in the other exons or in the promoter region of the gene either. The T + A  transversion introduces an AviII restriction site, a condition which allowed us to confirm the presence of the mutation in the proband’s genomic DNA and to screen the proband’s family members for the muta- tion. AviII digestion of exon 1 generates a single band of 235 bp in the control subjects and two additional bands (168 and 67 bp, respectively) in the proband (Fig. 2). None of the family members was found to carry the mutation. The absence of the mutation in the proband’s parents was also confirmed by direct sequencing of exon 1 (data not A ‘de ROVO’ point mutation of LDL-R gene Fig. 1.  Nucleotide sequence of exon 1 in a control subject ( C )  and in proband AN (FH-Enna). Proband AN is heterozygous for a T > A transversion which causes an arginine for tryptophane substitution at position - 12 of the signal peptide (W-12R) of the LDL-receptor. shown). Paternity testing was performed using a battery of 14 short tandem repeats located in 13 different chromosomes (Table 2). The result of this study defined a hypothesis of paternity with a casual sharing of 0.7 x lo-’. Discussion In this study we report a defect of the LDL-recep- tor function in a subject with primary hypercholes- terolemia carrying a ‘de nouo’ mutation in the LDL-receptor gene. To the best of our knowledge Fig. 2. Screening for FH-Enna mutation. Exon I was arn- plified by PCR. digested with the restriction enzyme AriII and separated on 2% agarose gel electrophoresis. The mutation introduces an AuiII restriction site in exon I. Lane 1. control subject; lane 2, proband’s father; lane 3, proband’s mother; lane 4. proband AN; lane 5 ,  proband’s brother. The size of the restriction fragments is shown on the left. this is the third report of a ‘de nouo’ mutation of the LDL-receptor gene associated with the clinical and biochemical phenotype of FH. The first ‘de nouo’ mutation (a duplication of 18 bp in exon 4) was reported in an FH-Afrikaner subject (8) and the second (a deletion of exons 14 and 15) was detected in a Finnish compound heterozygote (9). The fact that only three ‘Lie n o w ’  mutations of the LDL-receptor gene have been reported so far is rather surprising for two reasons: a) the large num- ber of mutations of this gene referred to in litera- ture (1); and b) the presence of several hot spots for mutations in this gene (CG dinucleotides, di- rect or inverted short repeats in coding sequences and the relatively large number of Alu sequences) (1, 10-12). It is possible that the apparently low rate of ‘de nouo‘ mutations of the LDL-receptor gene simply reflects a selection bias, as sporadic cases of primary hypercholesterolemia (especially in young subjects, as in our case) in the absence of a positive family history (tendon xanthomas, hy- percholesterolemia and /or premature CHD in first degree relatives) are misdiagnosed since they do not meet the clinical criteria usually adopted for the diagnosis of familial hypercholesterolemia. In view of our finding we suggest that the analysis of the LDL-receptor gene should be taken into con- sideration in all cases of sporadic primary h ypercholesterolemia. The mutation found in proband AN causes the substitution of tryptophane at position - 12 with arginine, a non-conservative amino acid change 393 Cassanelli et al. Table 2. Parental diagnosis ~ ~~ Chromosome Marker (STFis) Alleles Allele shared with father Father Mother Son 1 HumF13B 6 9  9/10 9 9  9 2 HurnTPOX 8 8  819 8 9  8 3 D3S1358 15 18 16,16 16 18 18 4 HurnFGA 21 23 23,24 21 23 21 5 HumCSF 11 11 11/12 11 12 11 6 D6S502 13 14 11/11 11 14 14 7 HurnF13AOl 5 6  3.216 5 6  5 8 HurnlPL 11 12 11/11 11 11 11 11 HumTHO1 6 8  919 69 9 6 12 HurnWVA31 A 17 17 1711 7 17 17 17 15 HurnFES FPS 10 11 11/12 10 11 10 18 D18S51 17 17 14/15 15 17 17 19 D19S253 12 13 12 14 13 14 13 21 D21Sll 63 68 61/63 61 63 63 which replaces an aromatic amino acid with a positively charged one. It is conceivable that the addition of an arginine residue at position - 12 of the signal peptide, by generating a stretch of posi- tively charged amino acids (Lys-Leu-Arg-Arg) up- stream from a long hydrophobic sequence, may impair the cotranslational-translocation of the nascent receptor protein across the membranes of the endoplasmic reticulum. The fact that the triplet Lys-Leu-Trp found at position - 14/ - 12 in the signal peptide of the human LDL-receptor is con- served in rat and hamster LDL-receptors suggests that this sequence may have an important role in co-translational translocation (1 3, 14). Co-transla- tional translocation of proteins across the mam- malian endoplasmic reticulum membrane involves several components such as the signal recognition particle receptor, the Sec61 complex and the translocation chain-associating membrane (TRAM) protein (15-18). It is generally assumed thit the features of the signal sequence, especially the length of its charged NH, terminal region and the length and the structure of the hydrophobic core, play a crucial role in the interaction of the nascent polypeptide chain with the translocation machinery. The efficiency of translocation derives from the balance between the positively charged residues of the N-terminal portion and the struc- ture of the hydrophobic chain (15). Although we do not have conclusive evidence that the W-12R mutation is the cause of the LDL-receptor defect in our proband, this mutation has never been found in the control subjects of our series, nor has it been reported by other investigators in nor- molipidemic subjects. Moreover, the same mutation (W-l2R), even if caused by a different nucleotide substitution (T > 394 C), has been recently detected in an FH coloured South African subject (1 9). Our ‘de nouo’ mutation was designated FH- Enna, from the city in Sicily where the proband’s family comes from. Acknowledgements This work was supported by a grant from the Italian Ministry of Education and Research (MURST) to SC and SB. References 1. Goldstein JL. Hobbs HH, Brown MS. Familial hyperc- holesterolemia. In: Scriver CR. Beaudet AL, Sly WS, Valle D, eds. The Metabolic and Molecular Bases of Inherited Disease. 7th edn. New York: McGraw-Hill. 1995: 1981- 2030. 2. Sambrook J, Fritsch EF. Maniatis T. Molecular Cloning. 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Hum Genet 1990: 85: 55-74. 11. Rideout WM, Coetzee GA, Olumi AF, Jones PA. 5- methylcytosine as an endogenous mutagen in the human LDL receptor and p53 genes. Science 1990: 249: 1288- 1293. 12. Krawczak M, Cooper DN. Gene deletions causing human genetic disease: mechanism of mutagenesis and the role of the local DNA sequence environment. Hum Genet 1991: 13. Lee LY, Mohler WA, Schafer BL. Freudenberger JS. Byrne-Connolly N, Eager KB, Mosley ST. Leighton JK, 86: 425-441. Thrift RN. Davis RA. Tmnka RD. Nucleotide sequence of the rat low density lipoprotein receptor cDNA. Nucleic Acid Res 1989: 17: 1259 -1260. 14. Bishop RW. Structure of the hamster low density lipo- protein receptor gene. J Lipid Res 1992: 33: 549-557. 15. Sakaguchi M. Tomiyoshi R. Kuroiwa T, Mihara K. Omura T. Functions of signal and signal-anchor sequences are determined by the balance between the hydrophobic segment and the N-terminal charge. Proc Natl Acad Sci 16. Corsi AK, Schekman R. Mechanism of polypeptide translocation into endoplasmic reticulum. J Biol Chem 17. Schatz G, Dobberstein B. Common principles of protein translocation across membranes. Science 1996: 271: 15 19- 1526. 18. Voigt S, Jungnickel B. Hartmann E, Rapoport TA. Signal sequence-dependent function of the TRAM protein during early phases of protein transport across the endoplasmic reticulum membrane. J Cell Biol 1996: 134: 25-35. 19. Marx MP, Thiart R. de Villiers NP, Kotze MJ. Spectrum of LDLR gene mutations in South Africa. Med Genet 1997: 9 (Suppl.): 161. USA 1992: 89: 16-19. 1996: 271: 30299-30302. 395 

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A ‘de novo’ point mutation of the low-density lipoprotein receptor gene in an Italian subject with primary hypercholesterolemia

A \u2018de novo\u2019 point mutation of the low-density lipoprotein receptor gene in an Italian subject with primary hypercholesterolemia

A \u2018de novo\u2019 point mutation of the low-density lipoprotein receptor gene in an Italian subject with primary hypercholesterolemia

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