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Journal of Clinical Lipidology (2018) -, -–-

1 52
2 53
3 54
4 Original Article 55
5 56
6
7 Screening of LDLR and APOB gene mutations 57
58
8 59
9 in Mexican patients with homozygous familial 60
10 61
11 hypercholesterolemia 62
12 63
13 64
14
Q7 s Hernandez Flores, MS, Juan Ramo
Teresita De Jesu n Gonzalez Garcıa, PhD, 65
15 Ana Gabriela Colima Fausto, MS, Norma Alejandra Vazquez Cardenas, PhD, 66
16 Yoaly Sanchez Lo
pez, PhD, C
esar Augusto Zarate Morales, MD, 67
17 68
18 ~a Torres, PhD*
Marıa Teresa Magan 69
19 70
20 71
Division de Genetica, Centro de Investigacion Biomedica de Occidente, Instituto Mexicano del Seguro Social,
21 72
Guadalajara, Jalisco, Mexico (Drs Hern andez Flores, Gonzalez Garcıa, Colima Fausto, Sanchez Lopez, and Maga~na
22 73
Torres); Doctorado en Genetica Humana, Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara,
23 74
Guadalajara, Jalisco, Mexico (Drs Hern andez Flores, Colima Fausto, Sanchez Lopez, and Maga~na Torres); Facultad de
24 75
Medicina, Universidad Aut onoma de Guadalajara, Guadalajara, Jalisco, Mexico (Dr Vazquez Cardenas); and Hospital
25 76
‘‘Presidente Ju
arez’’ del Instituto de Seguridad y Servicios Sociales de los Trabajadores del Estado (ISSSTE), Oaxaca,
26 77
Oax, Mexico (Dr Zarate Morales)
27 78
28 79
29 KEYWORDS: BACKGROUND: Familial hypercholesterolemia (FH) is an autosomal dominant disorder that causes 80
30 Familial accumulation of serum low-density lipoprotein cholesterol and premature cardiovascular disease. It is 81
31 hypercholesterolemia; mainly related to mutations in the LDLR gene. Homozygous FH (HoFH) patients have the most severe 82
32 Homozygote state; form of the disease accounting for a worldwide prevalence of 1:1,000,000. In Mexico, at least 5 cases 83
33 LDLR mutations; of HoFH have been reported. 84
34 Novel mutation c.- OBJECTIVE: The aim of this study was to describe the clinical, biochemical, and molecular data 85
35 139delCTCCCCCTGC observed in patients with HoFH phenotype. 86
36 METHODS: We included 13 patients, belonging to 11 families, with clinical and biochemical diag- 87
noses suggestive of HoFH. Molecular analyses of the LDLR and APOB genes were performed by
37 88
means of polymerase chain reaction followed by Sanger sequencing.
38 89
RESULTS: The causal mutation of HoFH was found in 8 of 11 unrelated patients. Excepting 1, all were
39 true homozygotes. Six different variants in LDLR were identified: c.-139delCTCCCCCTGC, p.Glu140Lys, 90
40 p.Asp360His, p.Asn405Lys, p.Ala755Glyfs*7, and p.Leu759Serfs*6. Of these, p.Asp360His and 91
41Q1 p.Asn405Lys were detected for the first time in Mexico; p.Leu759Serfs*6 showed to be the most frequent 92
42 (43.7% of the alleles 7/16), and c.-139delCTCCCCCTGC is a new variant located in the promoter region. 93
43 CONCLUSIONS: This work increases knowledge of biochemical and genetic features in Mexican 94
44 patients with HoFH. A novel mutation in the LDLR gene promoter was detected: 95
45 c.-139delCTCCCCCTGC, which possibly inhibits its expression. 96
46 Ó 2018 Published by Elsevier Inc. on behalf of National Lipid Association. 97
47 98
48 99
49 * Corresponding author. Centro de Investigacion Biomedica de Occi- E-mail address: maganamt@gmail.com 100
50 dente, Instituto Mexicano del Seguro Social, Sierra Mojada No. 800, Col. Submitted December 5, 2017. Accepted for publication February 24, 101
51 Independencia, Guadalajara, Jalisco, Mexico, C.P. 443400. 2018. 102

1933-2874/Ó 2018 Published by Elsevier Inc. on behalf of National Lipid Association.


https://doi.org/10.1016/j.jacl.2018.02.015

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2 Journal of Clinical Lipidology, Vol -, No -, - 2018

103 Introduction (33.3%) LDLR mutations have been detected exclusively 159
104 in Mexican patients.16–20 We here describe clinical, 160
105 Familial hypercholesterolemia (FH) is an autosomal biochemical, and molecular data of Mexican patients with 161
106 dominant disorder resulting in elevated serum low-density the HoFH phenotype. 162
107 lipoprotein cholesterol (LDLc) levels. The main clinical 163
108 manifestations include presence of planar, tuberous, or 164
109 tendon xanthomas; xanthelasma, corneal arcus, and prema- Material and methods 165
110 ture cardiovascular disease (CVD).1 Heterozygous FH 166
111 (HeFH) patients usually exhibit normal levels of triglycerides We studied 13 patients with the HoFH phenotype, 167
112 (TGs # 200 mg/dL) and an increase in both total cholesterol belonging to 11 families and from 5 different states of 168
113 (TC $ 300 mg/dL) and LDLc ($190 mg/dL).2,3 Homozy- the Mexican Republic (7 families from Oaxaca and 1 from 169
114 gous FH (HoFH) patients have the most severe form of the each of the following states: Baja California, Nayarit, 170
115 disease, which is characterized by appearance of the clin- Puebla, and Mexico City). Criteria to classify patients 171
116 ical symptoms during childhood, very high levels of TC with the HoFH phenotype were inherited familial 172
117 ($500 mg/dL) and LDLc ($400 mg/dL), as well as low background, TC value $ 500 mg/dL, and/or LDLc 173
118 high-density lipoprotein cholesterol (,40 mg/dL).4 The $ 400 mg/dL, as well as the presence of some clinical 174
119 worldwide prevalence in general population for HeFH features such as xanthomas, xanthelasma, corneal arcus, 175
120 and HoFH is 1:500 and 1:1,000,000, respectively.5 etc. This study was approved by our institutional ethics 176
121 However, in the Netherlands, a higher frequency of HoFH and research committees, and before blood sampling, all 177
122 patients has been reported (1:160,000–300,000).6 the participants signed an informed consent. For 178
123 Seventy-nine percent of patients with molecular diagnosis biochemical and molecular analyzes, each subject 179
124 of FH have mutations in the LDLR gene.7 The remaining provided 10 mL of blood, after 12 hours of fasting and 180
125 21% have mutations in the APOB,8 PCSK9,9 APOE,10 72 hours without alcohol intake. Plasma concentrations of 181
126 SREBP2,11 and STAP1 genes,12 although some cases could TC, high-density lipoprotein cholesterol, and TGs were 182
127 be due to hypercholesterolemia polygenic.7 Currently, 1718 quantified with the Architect C8000 chemistry analyzer 183
128 different mutations affecting the LDLR gene have been equipment using own kits. LDLc levels were calculated by 184
129 described: 1400 classified as pathogenic, 210 nonpatho- Friedewald equation21; however, they were quantified 185
130 genic, and 108 with unknown significance.13 directly only when the TG values exceeded 400 mg/dL. 186
131 Patients with HoFH can be categorized, according to Molecular analyzes of the LDLR and APOB genes Q2 187
132 their molecular genotype, as true homozygotes (patients were performed using genomic DNA obtained by a com- 188
133 who carry the same mutation on each allele), compound bined method (salting out and CTAB-DTAB).22,23 189
134 heterozygotes (patients who inherit a different mutation Twenty-one fragments of the LDLR gene, encompassing 190
135 from the same gene), and double heterozygotes (patients the 18 exons plus 250 bp of proximal promoter region, 191
136 carrying 2 mutated alleles from different genes).4 In were amplified by polymerase chain reaction (PCR) using 192
137 general, mutated alleles can be classified into 2 types: (1) primers previously reported.24 For APOB gene, we 193
138 null alleles, which are consequence of mutations in pro- analyzed a segment of 547 bp located in exon 26, which 194
139 moter regions, large rearrangements or deletions causing encodes a fragment of the ligand-binding domain and is a 195
140 frameshift, and premature stop codons and (2) defective hot spot where most of FH-related variants have been 196
141 alleles, which disrupt the binding, internalization, transport, found. Two pairs of primers designed with the Oligo 197
142 or recycling of the LDLc-LDLR complex.14 6.0 program were used. The first pair produces a fragment 198
143 The goal of pharmacological treatment is to reduce both of 325 bp (forward 50 CTTTCTCGGGAATATTCAG30 199
144 the serum LDLc levels and the cardiovascular risk. The and reverse 50 AATCATGGAAGGAACTGG30 ), and the 200
145 first-line therapy consists on the administration of statins, second one generates a fragment of 299 bp (forward 201
146 either alone or in combination with ezetimibe, which 50 CACCCTGGAACTCTCTCC30 and reverse 50 AATCC 202
147 increases the LDLR synthesis. Most patients with HoFH CATAAGCTCTTGTC30 ). PCRs were performed in a total 203
148 do not achieve sufficient reductions in LDLc levels even volume of 15 mL, containing 100 ng genomic DNA, 2.5 204
149 with the maximal doses of statins, making it necessary to pmol of each primer, 0.5 units of Taq chromo polymerase 205
150 supplement their treatment with either apheresis or liver (Vivantis), 0.2 mM of each dNTP, and 1.5 mM MgCl2. 206
151 transplantation.14,15 In Mexico, FH has been poorly studied Two different PCR conditions were used for the 23 frag- 207
152 and less than 1% of our population has been screened. To ments. Exons 1, 2, 4, and 7 of the LDLR gene were ampli- 208
153 date, 520 patients (including 5 HoFH) from 111 families fied as follows: 3 minutes at 94 C, 30 cycles of 30 seconds 209
154 have been reported, and the causal mutation was identified at 94 C, 30 seconds at 57 C, 45 seconds at 72 C, and 210
155 in 48.7% (54/111).16–20 In total, 33 causative mutations of finally, 7 minutes at 72 C. The remaining LDLR gene frag- 211
156 FH (31 in LDLR and 2 in APOB genes) were found, and the ments and the 2 APOB segments were amplified with a 212
157 most common LDLR variants were p.Cys352Tyr touch down program at 3 different temperatures: 60 C, 213
158 (FH-Mexico 2) and p.Cys364Arg (FH-Mexico 3), with 58 C, and 56 C. Amplified fragments were sequenced in 214
9% frequency each. It is important to highlight that 11/33 both directions using the Ready Reaction Big Dye

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Hernandez Flores et al LDLR mutations in Mexican homozygous patients 3

215 Terminator Kit v.3.1 (Applied Biosystems). Sequencing Mutational analysis 271
216 reactions were performed according to the protocol provided 272
217 by the manufacturer, and capillary electrophoresis was carried Molecular diagnosis was first performed in 11 unrelated 273
218 out on an ABI PRISM 310 Genetic Analyzer (Applied index cases, detecting the causal mutation of FH in 8 of 274
219 Biosystems). Sequences were analyzed with the Sequencing them: 7 were true homozygotes (87.5%), and 1 was a 275
220 Analysis Software 5.2 (Applied Biosystems), and electrophe- compound heterozygote (12.5%); in 3 patients, no mutation 276
221 rograms were reviewed with the 4Peaks software. Once the was found for the 2 analyzed genes (LDLR and APOB; 277
222 FH-causing variant in the index case was identified, relatives Table 1). In total, 6 different variants in the LDLR gene 278
223 were also tested for such specific mutation. were observed: (1) c.-139delCTCCCCCTGC, a novel mu- 279
224 In silico analyzes to predict the impact of variants on tation located in the promoter region; (2) p.Glu140Lys 280
225 stability, function, and structure of the protein were per- (c.418G.A, rs748944640) in exon 4; (3) p.Asp360His 281
226 formed with 6 online programs: (1) PolyPhen-225 (c.1078 G.C, rs777926251) in exon 8; (4) p.Asn405Lys 282
227 (benign 5 0 and damaging 5 1); (2) MutationAssesor26 (c.1215C.G; rs879254837) in exon 9; (5) p.Ala755- 283
228 (impact low-moderate-high); (3) Variant Effect Predictor Glyfs*7 (c.2264_2273delCCACCCCTGG; rs879255168) 284
229 (VEP)27 (impact low-moderate-high and modifier; http:// in exon 15; and (6) Leu759Serfs*6 (c.2271delT; 285
230 www.ensembl.org/Tools/VEP); (4) PROMO28 (shows the rs875989940) in exon 15. The most frequent mutation 286
231 presence or absence of consensus sites for different tran- was Leu759Serfs*6 (7/16 alleles, 43.7%), which was 287
232 scription factors; http://alggen.lsi.upc.es/cgi-bin/promo_ observed in 4 patients (3 true homozygotes and 1 com- 288
233 v3/promo/promoinit.cgi?dirDB5TF_8.3); (5) HOPE29 pound heterozygote); the remaining variants were detected 289
234 (The data obtained were classified as altered/nonaltered in a different family each (Table 1 and Fig. 1). After 290
235 amino acid properties and altered/nonaltered domain analyzing the 11 index cases, we focused on detecting the 291
236 tasks); and (6) CUPSAT30 (to assess changes in protein specific mutation in 23 relatives of the 8 patients with de- 292
237 stability and torsion angles; the results are given as tected mutations. In total, 21 relatives had mutations: 1 293
238 stabilizing/destabilizing structure and favorable/ true homozygote (II-1-family 3), 1 compound heterozygote 294
239 unfavorable torsion angles). (II-3-family 6), and 19 heterozygotes (Fig. 1). The clinical 295
240 and biochemical characteristics of the 2 patients with HoFH 296
241 (II-1-family 3 and II-3-family 6) are described in Table 1. 297
242 Results Variants found in the 19 heterozygote relatives were 298
243 p.Glu140Lys (n 5 2), p.Asp360His (n 5 4), p.Asn405Lys 299
244 Clinical and biochemical characteristics (n 5 2), p.Ala755Glyfs*7 (n 5 3), Leu759Serfs*6 300
245 (n 5 7), and c.-139delCTCCCCCTGC (n 5 1). As ex- 301
246 We analyzed 13 patients (7 women and 6 men) with the pected, homozygote patients for either p.Ala755Glyfs*7 302
247 HoFH phenotype; of which, 11 were unrelated (2 families or Leu759Serfs*6 variant (classified as null alleles) had 303
248 from the Oaxaca state had 2 cases with HoFH each). higher LDLc and TC plasma levels than patients carrying 304
249 Patients’ clinical, biochemical, and genetic data are shown the p.Asp360His or p.Glu140Lys mutations (considered 305
250 in Table 1. The average age of patients was defective alleles, Table 1); however, the patient with 306
251 17.5 6 15.7 years (range 4–42 years). Means of TC and p.Asn405Lys mutation—classified also as defective 307
252 LDLc levels were 711.9 6 305.7 mg/dL and allele—had similar lipid levels to those carrying null muta- 308
253 644.2 6 313.9 mg/dL, respectively. We obtained the clin- tion, which is inconsistent with literature reports.31–33 Still, 309
254Q3 ical features only from 10 HoFH patients. All of them studies regarding LDLR activity are required. 310
255 showed planar and tuberous xanthomas in skin, buttocks, 311
256 elbows, knuckles, and/or Achilles tendon since the first In silico analysis 312
257 decade of life; xanthelasma and corneal arcus were present 313
258 in 10% (1/10) and 30% (3/10) of the cases, respectively. All variants were subject to an in silico analysis, and 314
259 CVD was a representative feature in 91% (10/11) of the pa- different software was applied considering the type of mu- 315
260 tients. Although apparently all patients were following a tation (Table 2). The results revealed that the novel variant 316
261 pharmacological therapy, only 3 of them (cases II-2- c.-139delCTCCCCCTGC modifies the binding site for Sp1, 317
262 family 2, I-2-family 5, and III-1-family 11) could inform c-Myb, and c/EBPb transcription factors, which are 318
263 the drug and dose of their therapy (combination of atorvas- involved in the regulation of the LDLR gene expression 319
264 tatin, ezetimibe, and cholestyramine; Table 1). To our (36-38). The remaining 5 mutations (p.Glu140Lys, p.As- 320
265 knowledge, only patient III-1-family 11 received plasma- p360His, p.Asn405Lys, p.Ala755Glyfs*7, and Leu759- 321
266 pheresis, which significantly decreased his cholesterol Serfs*6) showed a deleterious effect on the protein, 322
267 levels (before plasmapheresis: TC 5 1651 mg/dL and which was evidenced by the results obtained by, at least, 323
268 LDLc 5 1601 mg/dL; after plasmapheresis: TC 5 one of the programs and corroborated with the information 324
269 327 mg/dL and LDLc 5 276 mg/dL). of the ClinVar database. 325
270 326

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4
Table 1 Clinical, biochemical, and genetic characteristics of patients with homozygous familial hypercholesterolemia
Family 1 2 3 4 5 6 7 8 9 10 11
Case II-7 †
II-2 ‡,ǁ
II-1 ‡
II-2 ‡
II-1 ‡
I-2 †,{
II-2‡,x
II-3 ‡,x
II-3 †
II-9 II-1 II-1 III-1ǁ Mean 6 SD
Origin Pue Nay Oax Oax Oax Mex Oax Oax Oax Oax Oax Oax B Cal
Recruitment ISSSTE# IMSS** ISSSTE†† ISSSTE†† ISSSTE†† IMSS‡‡ ISSSTE†† ISSSTE†† ISSSTE†† ISSSTE†† ISSSTE†† ISSSTE†† ISSSTExx
institution
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Age (y)* 42 7 8 7 4 39 16 11 10 12 11 14 7 17.5 6 15.7


F
Gender F F M F F F M M F F M M M (54%),M(46%)
TC (mg/dL) 456 709 762 722 734 684 555 437 752 733 501 559 1651 711.9 6 305.7
LDLc (mg/dL) 382 679 704 659 674 608 416 362 642 694 455 498 1601 644.2 6 313.9
HDLc (mg/dL) 26 28 27 38 26 39 35 36 30 17 27 27 19 29.5 6 7.3
TG (mg/dL) 236 - 156 124 171 185 104 161 196 100 172 172 154 160.9 6 38.4
Xanthoma Yes Yes – – – Yes Yes Yes Yes Yes Yes Yes Yes 10/10 (100%)
Xanthelasma No Yes – – – No No No No No No No No 1/10 (10%)
Corneal Arcus No Yes – – – Yes No No No Yes No No No 3/10 (30%)
CVD AI Noǁǁ – MI – SAS AS, AI MR AI AS, AI AI AS AH, AHC 10/11 (91%)
ITCA, AA
LDLR gene p.Asp360 p.Ala755 p.Leu759 p.Leu759 p.Leu759 p.Glu140 p.Leu759Serfs*6 p.Leu759Serfs*6 p.Asn405 p.Leu759 —— —— ——
Mutation His Glyfs*7 Serfs*6 Serfs*6 Serfs*6 Lys c.-139delCTCCC c.-139delCTCC Lys Serfs*6

Journal of Clinical Lipidology, Vol -, No -, - 2018


CCTGC CCCTGC
F, female; M, male; SD, standard deviation; TC, total cholesterol; LDLc, low-density lipoprotein cholesterol; HDLc, high-density lipoprotein cholesterol; TG, triglyceride; Pue, Puebla; Nay, Nayarit; Oax,
Oaxaca; Mex, Mexico City; B Cal, Baja California; ISSSTE, Instituto de Seguridad y Servicios Sociales de los Trabajadores del Estado; IMSS, Instituto Mexicano del Seguro Social; CVD, cardiovascular disease; AA,
aortic aneurysm; AH, aortic hypoplasia; AHC, apical hypertrophy cardiomyopathy; AI, aortic insufficiency; AS, aortic stenosis; ITCA, intimal-media thickening of the carotid artery; MI, myocardial infarction;
MR, mitral regurgitation; SAS, supravalvular aortic stenosis.
*Age at clinical and biochemical diagnosis.
†Defective mutation.
‡Null mutation.
xCompound heterozygote.
ǁ
Therapy: 20 mg atorvastatin, 10 mg ezetimibe, and 12 g cholestyramine.
{40 mg atorvastatin and 12 g cholestyramine.
#
Hospital Regional de Puebla-ISSSTE.
**Centro Medico Nacional de Occidente-IMSS, Guadalajara, Jal.
††Hospital ‘‘Presidente Juarez’’-ISSSTE,Oaxaca, Oax.
‡‡Hospital ‘‘La raza’’-IMSS, Mexico City.
xxHospital ‘‘20 de noviembre’’-ISSSTE, Mexico City.
ǁǁ
Patient not evaluated by a cardiologist, but apparently has not manifested any cardiological symptoms. Case III-1-family 11 had also been previously examined by sequencing for PCSK9, LDLRAP1, and
ABCG5/ABCG8 genes, as well as by Multiplex Ligation-dependent Probe Amplification for the LDLR gene, but no mutation was identified.
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Hernandez Flores et al LDLR mutations in Mexican homozygous patients 5

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474 Figure 1 Family pedigrees from the 11 studied families. In families 1–8, mutations related with the homozygous familial hypercholes- 530
475 terolemia phenotype were detected. Meanwhile, no mutation was found in families 9–11. Individual II-4-family 8 died having clinical char- 531
476 acteristics compatible with homozygous familial hypercholesterolemia. Y, years. The values of total cholesterol (TC) and LDLc are given in 532
477 mg/dL. 533
478 534
479 535
480 Discussion reported in patients from Mexico (21 6 13 years, 536
481 P 5 .73)17 and India (13.8 6 11.6 years, P 5 .48).34 Clin- 537
482 ical features of patients with HoFH are usually evident 538
Clinical and biochemical characteristics
483 from the first decade of life; however, 2 of our patients 539
484 were diagnosed after the third decade, probably due to a 540
FH is a genetic disease caused primarily by mutations in
485 lack of access to health systems and/or limited knowledge 541
the LDLR gene, which encodes for a transmembrane glyco-
486 of the disease in our country. LDLc levels reported in pa- 542
protein crucial in cholesterol homeostasis. HoFH patients
487 tients with HoFH have been very heterogeneous, ranging 543
have an extremely high risk of premature CVD. Although
488 from 400 to 1000 mg/dL.35 Such heterogeneity could be 544
the frequency of this very serious phenotype is unknown
489 related to the type of mutation causal of the disease and 545
in Mexico, it is estimated that there are more than 120 cases
490 to several variants located in other genes (MTTP, APOE, 546
with this condition.16–20 To our knowledge, 5 HoFH cases
491 APOB, LPL, LDLRAP1, APOA5, and ANGPLT3/4) that 547
(all them with true homozygote genotypes) have been re-
492 modulate the LDLc levels.36–38 This variability is also 548
ported in Mexican population.16–18
493 observed in our patients. 549
In our study, the mean age of HoFH detection was
494 We quantified TGs in 12 of the 13 HoFH patients 550
17.5 6 15.7 years (Table 1), similar to those previously
observing high TG levels (.150 mg/dL) in 9 of them

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6 Journal of Clinical Lipidology, Vol -, No -, - 2018

551 (Table 1). Although several studies have reported that sub- 607

Altered aa properties/altered

Altered aa properties/altered

Altered aa properties/altered
552 jects with this pathology present an increased rate of VLDL 608
553 apoB production, which would in turn increase TGs in 609
554 serum, other works have failed to demonstrate such effect. 610
555 Such discrepancies may be due to other factors, as the type 611

domain tasks

domain tasks

domain tasks
556 of mutation in the LDLR gene, polymorphic variants of 612
557 other genes (APOE, APOB, etc.), age, gender, or resistance 613
558 to insulin, among other factors.39 614
HOPE

559 Xanthomas are the most common clinical feature previ- 615


560 ously reported in patients with HoFH, occurring in 50% to 616
561 100% of cases.6,17,34,40,41 In this study, all patients with 617
Destabilizing effect/unfavorable

Destabilizing effect/unfavorable

562 clinical information (10/10) exhibited xanthomas, but xan- 618


Stabilizing effect/favorable

563 thelasma and corneal arcus were observed in 1 and 3 cases, 619
564 respectively. The latter 2 characteristics are less frequent 620
565 and/or have been poorly documented. Only patient II-2- 621
torsion angles

torsion angles

torsion angles

566 family 2 presented simultaneously the 3 clinical features 622


567 (Table 1). Both our results and some other reports make 623
568 evident the clinical variability usually observed in HoFH 624
CUPSAT

569 patients.34 625


570 Although apparently all our patients were under phar- 626

571 macological treatment, none of them showed near-normal 627


C/EBPb†

572 628
In silico predictions of mutations detected in patients with homozygous familial hypercholesterolemia

levels of LDLc. However, it is well known that patients


PROMO

c-Myb†
SP1†

573 with HoFH do not reduce their LDLc levels to optimal 629

574 values with conventional pharmacological treatment. Alter- 630


575 natively, new treatments have been developed focusing on 631
High effect/pathogenic

High effect/pathogenic
Moderate effect/likely
Moderate effect/likely

576 regulating the functioning of proteins engaged in lipid 632


577 metabolism, such as MTTP, APOB, PCSK9, and CETP.5,42 633
region variant

Moderate effect
TF binding site

578 Currently, anti-PCSK9 monoclonal antibody treatment is 634


pathogenic

pathogenic

579 available in Mexico43,44; however, information concerning 635


580 the type of patients being treated and the results of such 636
aa, amino acid; MA, MutationAssesor; VEP, Variant Effect Predictor; TF, transcription factor.

581 treatment is not yet available. The most extreme approaches 637
VEP

582 for HoFH patients are plasmapheresis or liver transplanta- 638


583 tion, which turn out to be not only expensive but also 639
Medium impact

584 mostly unavailable in our country. 640


High impact

Low impact

†Binding sites for these transcription factors were affected by the mutation.

585 641
586 Mutational and in silico analysis 642
587 643
MA

588 The causal mutation of FH was found in 8 unrelated 644


Software used

589 patients: 7 true homozygotes (87.5%) and 1 compound 645


damaging*

damaging*
PolyPhen-2

590 heterozygote (12.5%), who is the first patient in Mexico 646


*Previously reported by Leiden Open Variation Database.
Probably

Probably

591 harboring 2 different variants in the LDLR gene causing a 647


Benign

592 phenotype HoFH. Similar frequencies have been found in 648


593 Spaniard population with 85.7% (24/28) of true homozy- 649


594 gotes and 14.3% (4/28) of compound heterozygotes.41 650
Pathogenic/likely

Pathogenic/likely
ClinVar database

595 Nevertheless, in Dutch population, a balanced frequency 651


pathogenic

pathogenic

596 between both genotypes (50%) has been detected.6,40 These 652
Pathogenic

Pathogenic

597 differences could be a consequence of the diversity of mu- 653


598 tations occurring in each population, as well as migration 654
599 and nonrandom mating. 655

600 We did not detect LDLR or APOB gene mutations in 2 656


c.-139delCTCCCCCTGC

601 index cases (families 9 and 10), although their lipid profiles 657
p.Leu759Serfs*6
p.Ala755Glyfs*7

602 and inheritance patterns were consistent with HoFH diag- 658
rs748944640

rs777926251

rs879254837

rs879255168

rs875989940
p.Asn405Lys
p.Asp360His

603 nosis (Table 1). It is well known that around 8% of muta- 659
p.Glu140Lys

604 tions causing the disease are due to large rearrangements 660
Table 2

Variant

605 in the LDLR gene.13 Unfortunately, such large rearrange- 661


606 ments cannot be detected by the methods used in our study. 662
Therefore, analysis of the LDLR gene through the

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Hernandez Flores et al LDLR mutations in Mexican homozygous patients 7

663 Multiplex Ligation-dependent Probe Amplification tech- required for the binding of APOB and APOE proteins.48 719
664 nique could be a good approach. Moreover, other genes The p.Glu140Lys variant was previously reported in several 720
665 such as PCSK9, APOE, SREBP2, and STAP1 should also populations: Mexican,17,24 Spanish,49 Japanese,50 721
51 52 53 54
666 be studied. Korean, Danish, Indian, and Italian. This mutation 722
667 On the other hand, lipid profiles of relatives of the occurs in a CpG dinucleotide,17 which is a potentially hy- 723
668 patient III-1-family 11 were normal, suggesting an auto- permutable motif because, at least, 4 other different muta- 724
669 somal recessive inheritance pattern, which led us to analyze tions (p.Glu140Asp, p.Glu140*, p.Glu140Gly, and 725
670 the LDLRAP1 gene. Because no mutations were found p.Glu140Lys) have been detected in this position. 726
671 there, we turned to the LDLR, APOB, PCSK9, ABCG5, In vivo assays of the p.Glu140Lys mutation have re- 727
672 and ABCG8 genes, finding no mutations in them, either. vealed 15%–30% of receptor activity.53 This diminution 728
673 These results make it necessary to search for mutations in could be a consequence of a destabilizing effect and of un- 729
674 the APOE, SREBP2, and STAP1 genes, or to suspect favorable torsion angles, as inferred by CUPSAT software. 730
675 some other pathology related to the genetic condition in Furthermore, we detected by HOPE program a possible 731
676 this family. repulsion and destabilization in the ligand-binding domain 732
677 because the mutant amino acid (residue 140) alters the cal- 733
678 New variant c.-139delCTCCCCCTGC cium binding site, preventing the formation of both 734
679 hydrogen bonds (with histidine 129 and serine 130) and 735
680 Six different mutations were detected in 8 index cases: 5 salt bridges (with leucine 14 and glycine 48). 736
681 of them have been previously reported and 1 is a novel 737
682 mutation. This new variant, c.-139delCTCCCCCTGC, is a p.Asp360His 738
683 10-bp deletion located in the promoter region of the LDLR 739
684 gene and was observed in the index case II-2-family 6. The p.Asp360His (c.1078G.C) mutation is categorized 740
685 DNA family analysis revealed the presence of this mutation as a defective allele, and it has been observed only in 741
686 in the mother and in the younger brother (Fig. 1). It is Japanese population.55 It is located in the epidermal growth 742
687 important to emphasize that the mother showed slightly factor (EGF)–like precursor domain (specifically in subdo- 743
688 elevated cholesterol levels (TC 5 243 mg/dL and main B).56 Noteworthy, another variant (p.Asp360Tyr) also 744
689 LDLc 5 150 mg/dL). This mild hypercholesterolemia associated to FH was found at the same position.55 745
690 seen in the mother could be the result of a phenomenon According to in silico analysis, the variant p.Asp360His 746
691 of incomplete penetrance, which has been detected in about showed an impact prediction as low, moderate, and benign 747
692 10% of patients with FH.16 Interestingly, the c.-139C.G with software PolyPhen-2, MutationAssesor, and VEP, 748
693 LDLR variant (involving the change of a single base) was respectively. However, 2 programs predicted modifications 749
694 observed in a HeFH patient with very high cholesterol at protein level, with the presence of histidine in residue 750
695 levels (TC 403.9 mg/dL and LDLc 317.6 mg/dL).45 360: CUPSAT revealed a destabilizing effect and unfavor- 751
696 In silico analysis of the mutation c.- able modifications in torsion angles of the protein, and 752
697 139delCTCCCCCTGC with the PROMO software HOPE set it as a possible modifier of interactions between 753
698 (Table 2) showed alteration in the DNA binding sites for EGF domain and other molecules/domains. With these pre- 754
699 3 transcription factors: SP1, c-EBPb, and c-Myb. The dictions, we can assume that the p.Asp360His mutation 755
700 variant removes 10 bp from the SP1 factor binding site could increase the degradation rate of LDLR by 2 mecha- 756
701 (2143 to 2130), which is very important for the transcrip- nisms: (1) favoring the binding of LDLR to PCSK9 or 757
702 tion of the LDLR gene. In this segment, at least 9 mutations (2) altering the conformational structure formed by EGF- 758
703 have been described (c.-142C.T, c.-139C.A, c.-139C.G, B, EGF-A, and LA7 subunits, which is necessary to protect 759
704 c.-138delT, c.138T.C, among others). Functional analyzes LDLR when it is inside the endosome.57,58 We, therefore, 760
705 of some of them have revealed significant reduction in the suggest that the p.Asp360His mutation is a causative 761
706 LDLR gene expression (range of 74% to 95%).13 On the variant of FH, but an in vitro assay is necessary to elucidate 762
707 other hand, EBPb and c-Myb activate alternate transcrip- the functional effect of such variant. 763
708 tion pathways of the LDLR gene, which means that the 764
709 presence of the mutation could affect its expression.46 p.Asn405Lys 765
710 Therefore, we suggest that the novel mutation c.- 766
711 139delCTCCCCCTGC causes FH, although functional The p.Asn405Lys (c.1215C.G) mutation has been 767
712 studies are required to confirm such hypothesis. previously described in South African patients.59 It is 768
713 located in a b propeller sheet segment, in the EGF-like pre- 769
714 p.Glu140Lys cursor domain.13 In silico assays with MutationAssesor and 770
715 CUPSAT revealed that the p.Asn405Lys variant is 771
716 LDLR mutation p.Glu140Lys (c.418G.A) is classified nonpathogenic. However, analysis with HOPE software 772
717 as a defective allele and is located in the third repeat of showed that this mutation is pathogenic because it could 773
718 the 7 that conform the LDLc binding domain of LDLR.47 affect the doubling of the b propeller subunit, since the 774
This change modifies the triad (Ser-Asp-Glu) that is size difference between asparagine (wild-type) and lysine

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8 Journal of Clinical Lipidology, Vol -, No -, - 2018

775 (mutant) residues causes the loss of a hydrogen bond be- in the bibliographic search, elaboration of the figure, and 831
776 tween 405-asparagine and 647-leucine residues. It has discussion of results. A.G.C.F. was involved in laboratory 832
777 been suggested that the b propeller subunit exerts an autor- activities. N.A.V.C. and C.Z. remitted patients and/or blood 833
778 egulation system, by interacting and releasing the ligand samples, and they made clinical and biochemical diagnosis 834
779 (LDLc) from the LDLc-LDLR complex in the endo- of HoFH individuals. M.T.M.T. designed the study and 835
780 some.58,60 A mutation causing an abnormally stable bind- wrote the manuscript. She confirmed diagnosis of HoFH 836
781 ing of the b propeller subunit with the ligand-binding patients, conducted laboratory assignments, and analysis of 837
782 domain would result in an increase of cholesterol levels results. All authors read, approved, and agreed with the 838
783 in blood. material contained in this manuscript. 839
784 840
785 p.Ala755Glyfs*7 and p.Leu759Serfs*6 841
786 Financial disclosure Q6
842
787 Q4 The p.Ala755Glyfs*7 mutation consists of a 10-bp dele- 843
788 tion causing a frameshift, a stop codon 7 codons down- All authors stated that no conflicts of interest exist. 844
789 stream and, consequently, a truncated protein. On the 845
790 other hand, the variant p.Leu759Serfs*6 results from a 846
791 deletion of T in nucleotide c.2271, producing a frameshift References 847
792 deviation and a stop codon. It is the most frequent mutation 848
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