Namrata Gupta, Rossella Spina, Patrizia Tarugi, Sekar Kathiresan and Maurizio R. Averna
Angelo B. Cefalù, James P. Pirruccello, Davide Noto, Stacey Gabriel, Vincenza Valenti,
Liver Cancer, and Hypocholesterolemia
Mutation Identified by Exome Sequencing Cosegregates With Steatosis,
Print ISSN: 1079-5642. Online ISSN: 1524-4636
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2013;33:2021-2025; originally published online May 30, 2013;
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at UniversitÃ degli Studi di Milano on January 6, 2014
plasma levels of total cholesterol, low-density lipoprotein-
cholesterol (LDL-C), and apolipoprotein B (apoB) below the
fifth percentile of the distribution in the population.1,2 Familial
HBL (FHBL; OMIM 107730) is the most frequent monogenic
form of HBL. It may be attributable to loss-of-function muta-
tions in APOB or, less frequently, in PCSK9 genes.1–5
The best-characterized form of FHBL occurs with domi-
nant inheritance (≈50% of FHBL) and has been linked to
heterozygous pathogenic mutations in the APOB gene.1 Most
APOB gene mutations lead to the formation of truncated apoB
protein of various sizes which, to a variable extent, lose the
capacity to form plasma lipoproteins in liver and intestine and
to export lipids from these organs.1,2 Missense nontruncating
mutations of the APOB gene can also be the cause of FHBL6,7
and are associated with a decreased secretion of the mutant
ypobetalipoproteinemia (HBL) represents a hetero-
geneous group of disorders characterized by reduced
apoBs because of an increased binding to microsomal triglyc-
eride transfer protein.6–8
As a consequence of impaired hepatic export of lipopro-
teins (very-low-density lipoprotein), subjects with FHBL
attributable either to truncating or nontruncating mutations
of the APOB gene are prone to hepatic steatosis.9,10 In these
subjects, the presence of fatty liver has been documented by
abdominal ultrasound examination, magnetic resonance, or
liver biopsy.9,11–14 Anecdotal reports have documented an asso-
ciation between fatty liver and steatohepatitis, liver cirrhosis,
and hepatocarcinoma in patients with FHBL.15–17
We studied a large family in whom we observed an autoso-
mal dominant pattern of low plasma cholesterol cosegregating
with fatty liver and hepatocarcinoma. We hypothesized the
presence in this family of a genetic susceptibility for cancer,
which cosegregates with a causal mutation of FHBL. To iden-
tify the causal mutation in this family, we performed exome
© 2013 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org DOI: 10.1161/ATVBAHA.112.301101
Objective—In familial hypobetalipoproteinemia, fatty liver is a characteristic feature, and there are several reports of
associated cirrhosis and hepatocarcinoma. We investigated a large kindred in which low-density lipoprotein cholesterol,
fatty liver, and hepatocarcinoma displayed an autosomal dominant pattern of inheritance.
Approach and Results—The proband was a 25-year-old female with low plasma cholesterol and hepatic steatosis. Low
plasma levels of total cholesterol and fatty liver were observed in 10 more family members; 1 member was affected
by liver cirrhosis, and 4 more subjects died of either hepatocarcinoma or carcinoma on cirrhosis. To identify the causal
mutation in this family, we performed exome sequencing in 2 participants with hypocholesterolemia and fatty liver.
Approximately 22 400 single nucleotide variants were identified in each sample. After variant filtering, 300 novel shared
variants remained. A nonsense variant, p.K2240X, attributable to an A>T mutation in exon 26 of APOB (c.6718A>T)
was identified, and this variant was confirmed by Sanger sequencing. The gentotypic analysis of 16 family members in
total showed that this mutation segregated with the low cholesterol trait. In addition, genotyping of the PNPLA3 p.I148M
did not show significant frequency differences between carriers and noncarriers of the c.6718A>T APOB gene mutation.
Conclusions—We used exome sequencing to discover a novel nonsense mutation in exon 26 of APOB (p.K2240X)
responsible for low cholesterol and fatty liver in a large kindred. This mutation may also be responsible for cirrhosis and
liver cancer in this family. (Arterioscler Thromb Vasc Biol. 2013;33:2021-2025.)
Key Words: carcinoma, hepatocellular ◼ exome ◼ fatty liver ◼ hypobetalipoproteinemia, familial, 1
Received on: December 30, 2012; final version accepted on: May 13, 2013.
From the Dipartimento Biomedico di Medicina Interna e Specialistica, Università degli Studi di Palermo, Palermo, Italy (A.B.C., D.N., V.V., R.S., M.R.A.);
Center for Human Genetic Research (J.P.P., S.K.), and Cardiovascular Research Center (J.P.P., S.K.), Massachusetts General Hospital, Boston, MA; Program
in Medical and Population Genetics, Broad Institute, Cambridge, MA (J.P.P., S.G., N.G., S.K.); Department of Life Sciences, University of Modena and
Reggio Emilia, Modena, Italy (P.T.); and Cardiology Division, Massachusetts General Hospital and Harvard Medical School, Boston, MA (S.K.).
*These authors contributed equally.
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.112.301101/-/DC1.
Correspondence to Maurizio Averna, MD, Professor of Internal Medicine, Dipartimento Biomedico di Medicina Interna e Specialistica (DIBIMIS),
Policlinico Paolo Giaccone, University of Palermo, Via del Vespro 141, 90127 Palermo, Italy. E-mail email@example.com
A Novel APOB Mutation Identified by Exome Sequencing
Cosegregates With Steatosis, Liver Cancer, and
Angelo B. Cefalù,* James P. Pirruccello,* Davide Noto, Stacey Gabriel, Vincenza Valenti,
Namrata Gupta, Rossella Spina, Patrizia Tarugi, Sekar Kathiresan, Maurizio R. Averna
Clinical and Population Studies
2022 Arterioscler Thromb Vasc Biol August 2013
sequencing, an approach that allows the identification of all
the coding variants present in affected family members.
Materials and Methods
Materials and Methods are available in the online-only Supplement.
DNA samples were available from 16 members across 2 gen-
erations. Lipid profiles, including apoB levels, and clinical
characteristics of the family are presented in Table 1.
The proband (subject IV-5) showed low levels of total cho-
lesterol, triglyceride, and LDL-C; low LDL-C levels were
found in 9 more subjects of the family with a dominant trans-
mission mode of inheritance (Table 1; Figure). Moreover 7 of
10 subjects with low cholesterol levels showed fatty liver as
determined by liver ultrasonography (Table 1).
The 2 samples that underwent exome sequencing each yielded
8.25 gigabases of sequence, with an average depth of cover-
age of ≈250 reads per targeted base. Approximately 22 400
single nucleotide variants were identified in each sample, of
which 15 237 passed the previously described filters and were
shared by both samples. After removing variants also identified
in pilot 1 of the 1000 Genomes project, 1509 shared variants
remained. After removing variants identified in 3 unrelated
samples ascertained attributable to a non–LDL-C–related lipo-
protein phenotype (hyperalphalipoproteinemia), 300 novel
shared variants remained. Of these, 112 were synonymous, 177
were missense, 4 were nonsense, and 7 were at splice sites.
Only 1 nonsense variant was within ±300 kilobases of lead sin-
gle nuocleotide polymorphisms in genomic regions associated
with LDL-C in a recent large-scale genome-wide association
study18: p.K2240X attributable to an A>T mutation in exon 26
of APOB (c.6718A>T), which encodes apoB 48.8.
The search for shared variants in the 2cM region encompass-
ing the c.6718A>T mutation of the APOB gene also revealed
an A>C mutation in exon 1 of the RHOB (ras homolog gene
family, member B precursor) gene (c.244A>C, p.M82L).
The presence of the nonsense mutation in exon 26 (c.6718A>T,
p.K2240X) was confirmed in 3 independent polymerase chain
reaction amplifications and direct sequencing in the proband
(subject IV-5). The mother of the proband (subject III-13) was
found to carry the same mutation in the heterozygous state.
Sanger sequencing of the same APOB gene region of exon 26
encompassing the mutation performed in the other 14 avail-
able family members confirmed that the nonsense mutation
segregated with the low cholesterol trait (Figure).
Table 1. Clinical Characteristics, Plasma Lipids, and Apolipoprotein B
Type of Severe
II:1* NANANA NANA NA NA Hepatocarcinoma
II:5* NANANA NANANANANA
Type 2 diabetes mellitus
IV:8 3714163 6257 66,4 21.1No Not presentWT
ApoB indicates apolipoprotein B; BMI, body mass index; HDL-C, HDL-cholesterol; LDL-C, low-density lipoprotein-cholesterol; NA, not available; TC, total cholesterol;
TG, triglycerides; and WT, wild type.
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Cefalù et al Fatty Liver and Hepatocarcinoma in FHBL 2023
There were 16 individuals in the family who had both
plasma lipids phenotype and DNA available for genotyping.
Of these 16 individuals, 10 were affected on clinical grounds
(total cholesterol below the fifth percentile). All ten affected
carried the APOB nonsense mutation. Of the 6 individuals
who were unaffected (total cholesterol >fifth percentile), none
carried the APOB nonsense mutation.
The direct sequencing of the region of exon 1 of RHOB gene
encompassing the c.244A>C variant showed that besides sub-
jects III-13 and IV-15, 2 other family members were heterozy-
gous carriers of the variant p.M82L (II-16 and IV-7; Figure).
To predict the effect of this amino acid change on protein func-
tion, we performed in silico analyses by using different algo-
rithms: PolyPhen (www.bork.embl-heidelberg.de/PolyPhen/),
SIFT (http://sift.jcvi.org/), and Mutationtaster (http://www.
mutationtaster.org/). The PolyPhen and SIFT algorithms gave
comparable results, indicating that the p.M82L (Polyphen
score: 0.002, SIFT score: 0.11) amino acid substitution had a
benign effect, whereas the Mutationtaster software predicted
the p.M82L missense mutation to be damaging (Score: 15).
Association of PNPLA3 Single
Nuocleotide Polymorphism rs738409
(I148M) With Hepatic Steatosis
As shown in Table 2, there were no significant differences in
either PNPLA3 rs738409 minor allele (G) frequency allele or
genotype frequencies between carriers and noncarriers of the
c.6718A>T APOB gene mutation.
In the present study, we describe a large family in whom low
plasma cholesterol, fatty liver, and hepatocarcinoma cosegre-
gate in an autosomal dominant pattern. Using whole exome
sequencing, we discovered that a novel nonsense mutation in
exon 26 of the APOB gene (c.6718A>T, p.K2240X) segre-
gates with low lipids and the liver phenotypes.
A large number of APOB gene mutations truncating apoB
have been reported to be the cause of FHBL, and novel muta-
tions are continually being identified in subjects with FHBL.19
FHBL heterozygotes are generally asymptomatic but most of
them develop fatty liver.19 In fact, individuals heterozygous for
inactivating mutations in APOB show impaired very-low-den-
sity lipoprotein particle metabolism and have a 3-fold increase
in hepatic triglyceride relative to healthy individuals.10
Earlier in vivo turnover studies have shown that effective-
ness of lipid secretion from the liver depends on apoB length,20
implying that a variable amount of lipids might accumulate
in the hepatocytes of FHBL carriers of different truncated
apoBs. It was also suggested that fatty liver always develops
in FHBL carriers of short and medium-size truncated apoBs
(<apoB-48), whereas other additional environmental factors
are needed in carriers of longer apoB forms.11
However, more recent data have shown that there is no
evidence that the size of apoB truncation could be associated
with a different degree of liver impairment. For instance, in
the patients cohort studied by Sankatsing et al,21 hepatic ste-
atosis was not more severe in patients carrying short truncated
Table 2. PNPLA3 Single Nucleotide Polymorphism rs738409 (I148M) in Carriers and Noncarriers of the c.6718A>T APOB Gene
PNPLA3 GenotypePNPLA3 Allele Frequency
ValueC/C n (%) C/G n (%) G/G n (%)CG
c.6718A>T APOB gene mutation carriers
c.6718A>T APOB gene mutation carriers with fatty liver
Noncarriers of c.6718A>T APOB gene mutation6 2 (33)3 (50)1 (17)0.580.42 NS
NS indicates not significant.
III:5 III:7III:9III:6 III:8 III:10
Figure. Pedigree of the family with familial hypobetalipoproteinemia. The proband is indicated by an arrow. Half filled denote affected
subjects, carriers of the mutation K2240X of the APOB gene; and empty symbol, unaffected subjects. RHOB indicates ras homolog gene
family, member B precursor; and WT, wild type. ?The cholesterol phenotype is unknown. *Subjects died of hepatocarcinoma. #Subject
affected by liver cirrhosis.
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2024 Arterioscler Thromb Vasc Biol August 2013
apoBs not secreted into the plasma compared with carriers of
Even if fatty liver in FHBL has been considered, per se, a
benign condition, a potential evolution to more severe forms
of liver diseases, such as steatohepatitis, cirrhosis, or liver car-
cinoma, seems to be a relevant clinical issue. To date, only a
few case reports on the association between FHBL and severe
liver diseases have been published.
Lonardo et al17 described a case of hepatocarcinoma with-
out cirrhosis in a subject with FHBL attributable to a truncated
form of apoB. The liver histology in this patient revealed a
moderate degree of steatosis and fibrosis outside the hepa-
tocarcinoma lesion, and the authors speculated that environ-
mental factors (such as alcohol and smoking) could trigger the
evolution of fatty liver attributable to FHBL. More recently,
Bonnefont-Rousselot et al16 have described a patient with
FHBL and liver cirrhosis attributable to a truncated form of
apoB. The liver biopsy revealed typical hepatic cirrhosis with
irregular nodules and macrovesicular steatosis. In this case,
classical causes of fatty liver and cirrhosis were excluded by a
comprehensive clinical, biological, and histological work-up.
To our knowledge, our observation is the first description
of the co-occurrence of FHBL, fatty liver, cirrhosis, and liver
cancer. In particular, participants II:1, II:5, III:3, and III:11
died of hepatocarcinoma. Furthermore, in participant III:11,
the histology of the liver tumor revealed a rare finding of
fibrolamellar hepatocellular carcinoma. In fact, fibrolamellar
carcinoma is a rare primary malignant liver tumor with dis-
tinctive histology that usually affects adolescents and young
adults with a nearly even sex distribution, and most patients
have no identifiable liver disease secondary to chronic infec-
tion with hepatitis B virus or hepatitis C virus.22
An interesting question deals with the identification of fac-
tors that could elicit a progression of fatty liver attributable to
FHBL to cirrhosis and liver cancer. Although environmental
and lifestyle influences are well known and prevalent poten-
tial contributors of progression of fatty liver, other molecular
processes may contribute to this condition. Recently, variants
in genes affecting lipid metabolism, oxidative stress, insulin
resistance, and immune regulation could act as predisposing
factors to the development of hepatic steatosis and the devel-
opment of progressive liver injury.23,24 Among these, 1 genetic
variant that has consistently been associated in many indepen-
dent studies with nonalcoholic fatty liver disease is a missense
mutation (Ile148→Met148 [p.I148M]) in patatin-like phos-
pholipase domain–containing (PNPLA) 3 gene (PNPLA3).25
Moreover, this PNPLA3 variant is not only associated with
hepatic steatosis but also with nonalcoholic steatohepatitis and
cirrhosis, and these data provide strong molecular evidence of
the importance of genetic factors on the progression of fatty
liver to more severe forms of hepatic diseases.25–27 Among
the APOB mutation carriers, individuals with the PNPLA3
p.I148M variant in this kindred did not show a higher sus-
ceptibility for fatty liver, suggesting that in this family the
PNPLA3 gene does not act as a predisposing factor to the
development of hepatic steatosis and the development of pro-
gressive liver injury.
In an attempt to identify other genetic determinants that
could contribute to the progression of liver disease in this
family, we searched for shared variants in the 2cM region
encompassing the c.6718A>T mutation of the APOB gene.
This analysis revealed an A>C mutation in exon 1 of the
RHOB gene (c.244A>C, p.M82L).
Ras-homologous (Rho) small GTPases are involved in the
regulation of a variety of cellular processes, and recent stud-
ies further confirmed the role of the Rho proteins in cancer
by showing their involvement in cell transformation, invasion,
metastasis, and angiogenesis.28
In particular, RhoB has a tumor-suppressive role, including
inhibition of cell proliferation and induction of apoptosis in
several human cancer cells, and inhibition of tumor growth
in a nude mouse xenograft model.29 Furthermore, RhoB is
inducible by genotoxic stress, such as UV light, some growth
factors (transforming growth factor-β), and chemotherapeutic
drugs (cisplatin and 5-FU).30
The results of genotyping of the p.M82L RhoB variant
showed no cosegregation with the APOB gene mutation found
to be responsible for the FHBL phenotype. However, it is
interesting to note that the 4 carriers of the p.M82L variant are
also carriers of the c.6718A>T mutation of the APOB gene
and are all affected by fatty liver; in addition, 1 of them (sub-
ject III:16) developed cirrhosis.
The clinical and genetic findings from this large kindred
suggest that the complex relationship between APOB muta-
tions responsible for FHBL and the clinical and pathological
sequelae of fatty liver accumulation requires further mecha-
We are indebted to the patients and their family for their cooperation
in this study.
Sources of Funding
This work was supported by contract grants from the University of
Palermo (60% to M.R. Averna and A.B. Cefalù), Grant 2009-RLKXPF
PRIN 2009 from the Italian Ministry of Education, University and
Research (to M.R. Averna), the Fondazione Cassa di Risparmio di
Modena (to P. Tarugi); J.P. Pirruccello was supported by the Sarnoff
Cardiovascular Research Foundation. S. Kathiresan is supported by
a Howard Goodman Fellowship and a Research Scholar Award from
the Massachusetts General Hospital, the Donovan Family Foundation,
and R01 HL-107816 from the US National Institutes of Health.
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Familial hypobetalipoproteinemia is a genetic disorder characterized by lower than fifth percentile plasma levels of total cholesterol, low-
density lipoprotein-cholesterol, and apolipoprotein B (apoB). Most of the cases with familial hypobetalipoproteinemia are attributable to
mutations in APOB gene, leading to defective hepatic secretion of apoB-containing lipoproteins (very-low-density lipoprotein). This results
in an impaired export of triglycerides from the liver causing fat accumulation and hepatic steatosis. Few case reports have documented the
association of familial hypobetalipoproteinemia with steatohepatitis, liver cirrhosis, and hepatocarcinoma. Here, we describe a large kindred
in which a novel mutation of APOB gene (p.K2240X), identified by exome sequencing, cosegregates with hypobetalipoproteinemia, fatty liver,
and hepatocarcinoma in an autosomal dominant pattern. We also found a variant in a tumor suppressor gene (RHOB), but no cosegrega-
tion was found with the lipid and hepatic phenotypes. In addition, genotyping of the PNPLA3 p.I148M does not show frequency differences
between carriers and noncarriers of the APOB gene mutation.
at UniversitÃ degli Studi di Milano on January 6, 2014 http://atvb.ahajournals.org/Downloaded from
Material and Methods
FHBL patients (TC < 5th percentile) are routinely identified and studied in the Lipid Clinic
of our Center for Genetic Dyslipidemias at the AOUP “P. Giaccone”, University of Palermo,
Italy. Over the years we have collected a large number of patients who underwent a
clinical, biochemical and genetic evaluation. Among these, one FHBL family with a high
prevalence of associated hepatic diseases was selected for exome sequencing.
Study participants and pedigree
The proband (Figure 1 - subject IV:5) is a 25 year-old female of European ancestry who
first came to our attention at the age of 18. Her clinical history was unremarkable except
for low plasma TC and triglycerides (TG) detected in several occasions (86 mg/dl and 44
mg/dl respectively). Moreover, an ultrasound evaluation of the abdomen showed liver
The analysis of plasma lipids of family members showed low plasma levels of TC and TG
in several others. Noticeably in subjects who underwent to ultrasound evaluation, fatty liver
was observed (subjects III:1, III:7, III:11, III:13, III:15, III:16 IV:5, IV:6 and IV:7) and in
subject III:16 the diagnosis of liver cirrhosis was made after he suffered an acute
It was also reported that four more subjects (II:1, II:5, III:3 and III:11) died of either
hepatocarcinoma (III:3 and III:11) or carcinoma on cirrhosis (II:1 and II:5). In particular
subject III-11 was referred to have a long-lasting unexplained history of
hypocholesterolemia who died at the age of 58 from massive gastroesophageal bleeding
after he was diagnosed with a hepatocarcinoma with a histologic finding of fibrolamellar
Chronic infection due to hepatitis B virus (HBV) or hepatitis C virus (HCV) and alcohol
abuse (mean alcohol intake was < 15g/day) were excluded in all the studied subjects. For
family members who had died of hepatocarcinoma, the exclusion of either chronic viral
infection or alcohol abuse was made by analyzing the clinical records available and
information provided by the relatives.
Moreover, as far as possible, other potential causes of chronic liver diseases, including
hepatic disorders of iron and copper metabolism, were excluded by analyzing the clinical
Plasma Lipid analysis
Blood samples were collected after an overnight fast. Blood (10 mL) was collected into a
plain tube and a tube containing EDTA (1 mg/mL) to obtain serum and plasma,
respectively, and buffy coat by centrifugation at 3000 rpm for 15 min. Plasma TC, TG and
high-density lipoprotein cholesterol (HDL-C) were measured using standard enzymatic–
colorimetric procedures (Roche Diagnostics, Basel Switzerland) on a COBAS MIRA plus
auto-analyzer (Roche Diagnostics, Basel Switzerland). LDL-C was calculated by the
Friedewald formula. ApoB plasma levels were measured by immuno-nephelometry using a
dedicated kit (Radim, Rome, Italy) on a DELTA (Radim, Rome, Italy) instrument.
Ultrasound (US) examination of the liver was performed to assess for fatty liver.
All US examinations were performed by a single operator with experience in liver disease
ultrasonography for more than a decade.
The US of the liver was performed in the morning after a 10 h fasting using a realtime
apparatus with a 2–5 MHz multi-frequency or 3.5 MHz convex probe.
The fatty liver diagnosis was made in the presence of fine, packed high amplitude echoes
that confer the brightness to the liver (bright liver) and hepatorenal echo contrast
Fatty liver severity was scored as follows: grade 1 = increased echogenicity or bright liver
with normal visualization of diaphragm and intrahepatic vessel borders; grade 2 =
increased echogenicity with posterior beam attenuation, but with slightly impaired
visualization of the intrahepatic vessels and diaphragm; and 3 = marked increase in
echogenicity and marked posterior beam attenuation resulting in failure to demonstrate the
intrahepatic vessels, diaphragm, and posterior right lobe of the liver .
Two participants with hypocholesterolemia and liver steatosis (Subjects III:13 and IV:5)
underwent exome sequencing. DNA from the two selected individuals was sent to the
Broad Institute in accordance with protocols put in place by the institutional review boards
of the AOUP “P. Giaccone” at University of Palermo and the Broad Institute. The DNA was
subjected to solution hybrid selection using oligonucleotides synthesized on an Agilent
array  in order to isolate the exonic genomic DNA, which was then sequenced using the
Illumina HiSeq platform with 76-nucleotide paired-end reads. A total of 32,950,014 bases
were targeted for sequencing. The sequence data was mapped to HG19 using BWA
(which implements the Burrows-Wheeler transform) , and single nucleotide variants
were called using the UnifiedGenotyper module of the Genome Analysis Toolkit (GATK)
. The variants were then filtered as follows: (1) Phred-scaled probability that a
polymorphism exists at a site > 30; (2) ratio of variant quality score to number of reads > 5;
(3) nonreference allele present in >25% of reads; (4) maximum contiguous homopolymer
run of the variant allele in either direction on the reference < 5 bases; and (5) strand bias
(as described in  less than -0.10. The variants were then annotated with a custom
Polymerase chain reaction (PCR) and DNA sequencing
Genomic DNAs from all subjects were extracted from whole blood using the Wizard DNA
Purification System (Promega Italia, Italy). A partial region of exon 26 of APOB gene was
sequenced using the primer pairs for PCR amplification and the amplification conditions as
previously described . PCR fragments were purified with a commercial kit (Wizard PCR
Preps–DNA Purification System; Promega Italia, Italy) then sequenced directly in both
directions using BigDye Terminator Cycle sequencing kit 1.1 in a ABI 310 DNA sequencer
(Applera Italia, Italy) and the results were analyzed with the Seqed software (Applied
Biosystems, Warrington, UK).
The direct sequencing of the region of exon 1 of RHOB gene variant was sequenced as
described above by using the following primers: EX1F GGGCCAGGAGGACTACGA and
Genotyping for rs738409 (I148M) in PNPLA3 gene
Genotyping for PNPLA3 rs738409 was conducted in a StepOne Real time Apparatus (Life
Technology) by using a commercial genotyping assay (cat. C_7241_10, Life Technology).
The genotyping call was done with SDS software v.1.3.0 (ABI Prism 7500, Foster City, CA,
USA). Genotyping was conducted in a blinded fashion relative to subject characteristics.
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