Kanuri et al. BMC Medical Genetics(2020) 21:234
https://doi.org/10.1186/s12881-020-01176-x

RESEARCH ARTICLE

Open Access

Mice lacking global Stap1 expression do
not manifest hypercholesterolemia
Babunageswararao Kanuri1, Vincent Fong1, April Haller2, David Y. Hui2 and Shailendra B. Patel1*

Abstract
Background: Autosomal dominant familial hypercholesterolemia (ADH; MIM#143890) is one of the most common
monogenic disorders characterized by elevated circulatory LDL cholesterol. Initial studies in humans with ADH
identified a potential relationship with variants of the gene encoding signal transducing adaptor family member
protein 1 (STAP1; MIM#604298). However, subsequent studies have been contradictory. In this study, mice lacking
global Stap1 expression (Stap1−/−) were characterized under standard chow and a 42% kcal western diet (WD).
Methods: Mice were studied for changes in different metabolic parameters before and after a 16-week WD regime.
Growth curves, body fats, circulatory lipids, parameters of glucose homeostasis, and liver architecture were studied
for comparisons.
Results: Surprisingly, Stap1−/− mice fed the 16-week WD demonstrated no marked differences in any of themetabolic parameters compared to Stap1+/+ mice. Furthermore, hepatic architecture and cholesterol content in
FPLC-isolated lipoprotein fractions also remained comparable to wild-type mice.
Conclusion: These results strongly suggest that STAP1 does not alter lipid levels, that a western diet did notexacerbate a lipid disorder in Stap1 deficient mice and support the contention that it is not causative for
hyperlipidemia in ADH patients. These results support other published studies also questioning the role of thislocus in human hypercholesterolemia.
Keywords: STAP1, Autosomal dominant familial hypercholesterolemia, Familial hypercholesterolemia 4, B-cells,
Western diet, Fast performance liquid chromatography

Background
Familial hypercholesterolemia (FH) is a widely prevalentcongenital metabolic disorder characterized by substantially elevated circulatory low density lipoprotein cholesterol (LDL-C) and accelerated cardiovascular events [1, 2].
Autosomal dominant inherited hypercholesterolemia(ADH) accounts for a major proportion of the FH cases
worldwide with mutations most commonly reported in
low density lipoprotein receptor (LDLR), apolipoprotein B(APOB), and proprotein convertase subtilisin/kexin type 9(PCSK9) [3–5]. However, a number of patients classified* Correspondence: sbpatel@uc.edu
1
Division of Endocrinology, Diabetes and Metabolism, University ofCincinnati, Cincinnati, OH, USA
Full list of author information is available at the end of the article

as having familial hypercholesterolemia 4 (FH4), demonstrate the ADH phenotype despite having no mutations inthese genes [6]. Initial studies involving these patients
reported mutations in genes coding for signal-transducing
adaptor family member 1 (STAP1) [6–8].
STAP1 (MIM#604298), also called B-cell antigen receptor downstream signaling 1 protein (BRDG1) or stem cell
adaptor protein 1, was first discovered in immune cellswith the highest expression documented in appendix,
lymph nodes, and spleen [7]. The gene encoding STAP1
protein is located on chromosomal region 4q13.2 with 10
exons in the protein coding region [7]. Its relationship to
lipid homeostasis was first suggested in 2007 in a clinicalstudy finding a correlation between increased levels of
circulatory triglycerides and STAP1 gene expression in© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate ifchanges were made. The images or other third party material in this article are included in the article's Creative Commonslicence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
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The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to thedata made available in this article, unless otherwise stated in a credit line to the data.

Kanuri et al. BMC Medical Genetics(2020) 21:234

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leukocytes [9]. Later, in 2014, Fouchier and colleagues discovered that variants of the STAP1 gene were associatedwith the FH phenotype in a linkage analysis of a Dutch
family with FH4 and suggested robust support for genetic
causality [6]. Subsequent studies reported STAP1 genevariants observed in such FH patients also manifested
significant cardiovascular events [10–12]. However, otherstudies found no association between STAP1 variants
and hypercholesterolemia [13–16]. An analysis ofseven families with FH phenotype failed to observe
the co-segregation of four rare predicted pathogenic
variants of STAP1 [17]. Loaiza et al more recently
demonstrated no marked changes plasma lipid profilesof carriers of STAP1 variants compare to controls aswell as in a mouse model [18]. Therefore, we sought
out to understand the STAP1 role by using a mouse
knockout model, to ask if loss of STAP1 would leadto hyperlipidemia.
In this study, mice lacking global expression of Stap1
gene (Stap1−/−) were characterized for various metabolic
parameters on standard diet and after 16 weeks of western diet (WD) regime. The present work presents an independent characterization of Stap1−/− mice to thatstudied by Loaiza et al with some differences between
models and confirms the argument against STAP1 having a role in causing FH.

16–04–19-01). Terminal blood samples were obtained
under full inhaled isoflurane anesthesia. For all studies,
except where indicated, each group consisted of 6 mice,based upon prior data that show the power to detect
20% differences for the most variable procedure (OGTT)
where the Power needed is n = 5, but we included andextra mouse to ensure Power was not due to loss of amouse for any inadvertent loss. The investigators were
not blinded to the samples or mouse genotypes, as there
are no datasets collected which required subjective analyses. However, all mice were group housed and eachcage contained mice of both genotypes to minimize bias
and variation. Mice were sacrified by CO2 inhalation,
followed by cervical dislocation and bilateral thoracotomy.

Methods

Oral glucose tolerance test (OGTT)

Knockout mice and diets

For the OGTT experiment, the experimental mice were
initially acclimatized in the procedure room overnight
followed by 4 h fast period in the next day morning. Afterbaseline blood glucose measurements, 50% Dextrose (Cat#
D16–1; ThermoFisher) in 0.9% saline was administered via
oral gavage at a dose of 2 g/kg of body weight [24]. Timedependent changes in blood glucose were measured via tailvein bleeds at 0, 15, 30 and 60 min using Accu-Chek Nanoelectronic glucometer (Roche Applied Science, Indianapolis,
IN, USA).

Heterozygous Stap1 KO mice (Stap1+/−) on C57BL/6 N
background generated through reporter-tagged insertion
were imported from Wellcome Trust Sanger Center, via
EUCOMM [19–22]. The ‘knockout first’ tm1a (Stap1tm1a(KOMP)Wtsi) mutation bears the IRES:lacZ trapping
cassette and a floxed promoter-driven neo cassette
inserted into the intron of the gene in order to disrupt
the gene function [22]. Brother-sister matings of theheterozygotes generated homozygous knockout mice
(Stap1−/−) and corresponding WT (Stap1+/+) littermate
controls. Tail snip genotyping was performed to assess
the genotype of each mouse before recruiting into experiments; amplimers of 266 bp and 236 bp correspond to
Stap1+/+ and Stap1−/−, respectively (Table). Mice werefed with irradiated standard rodent-chow diet (CD;
Envigo 7912) [23] and water ad libitum, with equal lightand dark cycles. Baseline measurements were performedin 10 to 11 week-old male and female mice on rodent
chow and then placed on a high-cholesterol western diet
(WD; Envigo TD.88137) for another 16 weeks [24].
Chow and western diets provided 17 and 42% kcal energy from fats [23, 24] and were procured from Harlan
Teklad, Madison, WI, USA. All animal studies wereapproved by the Institutional Animal Care and Use
Committee at the University of Cincinnati (Protocol No.

Measurement of whole-body composition

Whole-body fat and lean mass were measured in live miceusing ECHOMRI™-100H whole body composition analyzer(Echo Medical Systems, Texas, USA) according to the
manufacturer’s specifications. Absolute weights (g) of fat
and lean mass of each mouse were used to calculate their %changes [(absolute fat or lean mass weight (g)/whole bodyweight(g))*100], a widely employed method to determine
the diet induced obesity phenotype.

Fast performance liquid chromatography (FPLC)

Lipoprotein separation for total cholesterol estimation inplasma samples was performed using FLPC as described
previously [24]. Briefly, blood was collected from 4 h
fasted mice through the submandibular plexus and thencentrifuged at 2500×g for 10 min at 4 °C. The isolated
plasma was then measured for basal total cholesterol to
exclude any outliers (to ensure pooling was performed between comparable samples) and 200 μL of pooled plasma
from each group was loaded onto the Akta pure FPLC toseparate different lipoproteins by column chromatography(10/300GL, superpose 6). A total of 52 fractions with volume of 500 μL each were collected and analyzed for cholesterol content using an enzymatic kit as detailed below.

Kanuri et al. BMC Medical Genetics(2020) 21:234

Plasma biochemistry

Plasma triglycerides and cholesterol were estimatedusing Infinity Triglyceride (TR22421) and Cholesterol(TR13421) calorimetric kits (Thermo Fisher Scientific,
Middletown VA, USA) per manufacturer instructions.
For estimation of tissue lipids after the western dietphase, livers from Stap1+/+ and Stap1−/− mice were
homogenized, normalized for equal protein content, andthen measured for total cholesterol and triglycerides
using the same calorimetric kits used for plasma samples.
Tissue harvesting and histology of liver

Mice were euthanized for terminal tissue collection at
the end of 16 weeks WD regime. Four-hour pre-fasted
mice were euthanized by CO2 exposure and bilateralthoracotomy. Liver, spleen, and kidneys were blotted
dry, weighed, and then flash frozen in liquid N2. A small
portion of liver was rinsed in chilled PBS (pH -7.4) and
fixed in 10% neutral buffer formalin (5725; Fisher Scientific, Pittsburg, PA, USA) for paraffin embedding andhematoxylin and eosin (H&E) staining [25]. About 5 μm
of paraffin embedded sections were cut, processed in
graded volumes of ethanol, and then stained with H&E.
Images of the stained liver sections were captured at 20x
and 40x objective lens magnification using Olympus
BX61 microscope.

Page 3 of 9transcript levels of Stap1 in WT mice by qPCR revealed
gene expression in a variety of tissues with the highest
levels demonstrated in spleen (Fig. 1b). The complete
absence of Stap1 expression was confirmed with qPCR
in Stap1−/− mice liver, kidney, and spleen (Fig. 1c). RTPCR analysis of exons 2–5, 2–6, and 2–7 using total
RNA isolated from livers and spleens demonstrated sequential exon splicing of exons 5, 6, and 7 in Stap1+/+
mice and no products in Stap1−/− mice (Fig. 1d). The
single slightly larger amplicon for exon 2–7 in Stap1−/−
spleen (Fig. 1d, open arrowhead) was sequenced anddemonstrated an off-target product on chromosome 2(Additional file 2).
Baseline characteristics

Baseline measurements on standard chow diet wereassessed at 10–11 weeks of age and body weights(Fig. 3a), whole-body fat (Fig. 2a) and lean mass (Fig. 2a)
were similar between Stap1+/+ and Stap1−/− mice.
Further biochemical assessment found no significant
differences in glucose tolerance (Fig. 2b), plasma totalcholesterol (Fig. 2c), or plasma triglycerides (Fig. 2d) between knockout mice and wildtype mice. Lipoproteincholesterol profiles in male knockout and wild-type mice
were analyzed by FPLC (Fig. 2e) and were also found to
be unaffected by the loss of Stap1.

QPCR analysis of gene expression

Effects of western diet (WD)To determine the tissue expression profile of Stap1, total
RNA were isolated from different tissues of Stap1+/+ miceusing Qiagen RNeasy Mini Kit (74,104; Qiagen Inc., Germantown, MD, USA). Isolated RNAs were reverse transcribed to cDNA using SuperScript First-Strand Synthesis
kit (11904–018; Invitrogen, Carlsbad, CA, USA) and thenassessed for the Stap1 gene expression on Applied Biosystems 7300 Real-Time PCR system (Applied Biosystems,
Foster City, CA, USA). Each sample was run in triplicatesusing power SYBR Green PCR master mix (4,367,659; Applied Biosystems, Foster City, CA, USA). Comparative CT
method was used for quantitation post data normalization
to Atp5po expression as the house keeping gene to compensate the variations between input RNA amounts [25].
The list of different primers used for PCR experiments
was tabulated in the Table S1 (Additional file 1).

To determine the role of Stap1 under metabolic
challenge, mice were fed western diet (42% kcal fromfat). Prior to the commencement of the western diets,
baseline body weights showed no statistical differences
between wildtype and knockout mice (Stap1+/+ males
25.2 g +/− 3.2 vs Stap1−/− males 23.5 +/− 3.1, Stap1+/+
females 22.5 +/− 2.6 vs Stap1−/− females 19.7 +/− 1.3,
n = 6 for each group). Body weights were measured
weekly (Fig. 3a), while body composition (Fig. 3b) andtissue weights (Fig. 3c and d) were assessed the end of
the WD. No significant differences were noted between
Stap1−/− mice over Stap1+/+ littermate controls in any ofthese parameters. Similar trends were also observed inglucose tolerance testing, monitored to ensure there
were no differential changes in intermediary metabolism
induced by the western diet and loss of Stap1 (Fig. 3e).
Plasma cholesterol and triglyceride levels (Fig. 4a andb), and plasma FPLC cholesterol profiles (Fig. 4c and d)showed the diets led to a significant change in the lipoprotein distribution from baseline (compare to Fig. 2e)as expected for a high fat diet, but there were no differences between wild-type and Stap1 knockout mice.
Investigation of liver histology with hematoxylin andeosin staining (H&E) showed changes compatible with
fat accumulation as would be expected after 16 weeks ofWD, but found no differences in morphology between

Statistical analyses

An unpaired t-test (without or without Welch’s corrections)or Mann-Whitney U test was performed to assess significant
between Stap1−/− and Stap1+/+ mice within each sex.

Results
Validation of Stap1 gene deletion

The targeting strategy for generation of global Stap1
knockout mice is depicted in Fig. 1a. Measurement of

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Fig. 1 Verification of global deletion of Stap1 gene expression. Panel a shows the gene targeting details for the generation of Stap1−/− mice. This
construct is a ‘knockout-first’, which can be converted to a conditional, upon deletion of the LacZ-Neo cassette. The mice used in this study
remain as knockout and retain this cassette. A tissue survey of Stap1 mRNA was performed using qPCR in tissues collected from Stap1+/+ mice(panel b, n = 2). Note that compared to a house-keeping gene expression (see Methods), only spleen and lung showed some expression withvery low levels detected in other tissues. Stap1−/− mice (KO) showed no detectable qRT-PCR products (panel c, n = 2–3) compared to wild-typemice (WT) in spleen, liver or kidney. To further confirm a lack of legitimate transcript expression, RT-PCR analysis of exon splicing for exons 2–5,
2–6, and 2–7 in liver and spleen was performed (panel d). The labels ‘5, 6, and 7’ for each tissue indicates the location of the reverse primers
targeting exons 5, 6, or 7 respectively, used with a forward primer located in exon 2. Spleen mRNA from WT tissues amplified the correct
expected size of product and size increments (exon 2–5; 251 bp, exon 2–6; 482 bp, and exon 2–7; 536 bp indicated by filled arrowheads). No
products were noted when exons 2–5, or 2–6 were used for KO spleen mRNA. A slightly larger product was noted for exons 2–7 (indicated byopen arrowhead), though on sequencing this was found to be a non-specific product from mis-priming (see Text). Error bars denote ±1 SD

groups (Fig. 5a and b). Moreover, the elevated levels of
liver total cholesterol and triglycerides (Fig. 5c and d) werecomparable between WD fed Stap1+/+ and Stap1−/− mice.

Discussion
This study tested the hypothesis that loss of Stap1 canlead to hypercholesterolemia using Stap1−/− mice. The
Stap1 mice, targeted by a knockout-first, conditional targeting vector were obtained from the Welcome Trust
Sanger Center and characterized. Although there is no
reliable antibody for specific detection of endogenous
STAP1 protein by western blot (we tested commercial
antibodies from ThermoFisher and Abcam but foundthese unsuitable, see Supplementary files), a knockout
was confirmed at the DNA and mRNA level instead.
Normal Stap1 RNA expression observed in different

tissues of Stap1+/+ mice and compared with the proteinatlas database (https://www.proteinatlas.org/ENSG0000
0035720-STAP1/tissue); relatively high levels of expression in spleen were noted but very minimal expression
was seen in liver. The higher levels of RNA also coincidewith the proportional amount of STAP1 protein in
spleen of Stap1+/+ mice as measured through targeted
proteomics [18]. QPCR analysis of livers, spleens, andkidneys of Stap1−/− mice did not detect any Stap1
mRNA, confirming the knockout (Fig. 1c). RT-PCR analysis of the mRNA produced expected amplicons whenusing primers targeted to exon 2 through 4 in Stap1−/−
mice (data not shown), but not with primers specific forexons 5, 6 or 7 (Fig. 1d) suggestive that the transcriptional message was preserved until exon 4 and was interrupted due to reporter insertion in the intronic region

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Fig. 2 Baseline metabolic parameters in chow-fed Stap1−/− and control mice. Panel a shows the starting body composition of mice at age 5–6
weeks on a chow diet (n = 6–7). Starting lean or fat masses between wildtype and knockout mice were comparable. Glucose tolerance (Panel b,n = 6), plasma cholesterol (Panel c, n = 6), triglycerides (Panel d, n = 6) were also comparable and showed no statistical differences. Panel e shows
the FPLC cholesterol profiles in 10–11 weeks old male Stap1+/+ and Stap1−/− mice and was also indistinguishable. Note that the knockout mice
do not show any differences in the VLDL (fractions 3–6), or LDL (fractions 20–28) ranges. Error bars denote ±1 SD

between exon 4 and 5. RT-PCR using primers targeted
to exon 2 through 7 unexpectedly produced a product in
Stap1−/− mice (Fig. 1d), but sequencing showed that it
was an off target product from chromosome 2 (supplemental data), whereas Stap1 is located on chromosome 4.
Obesity and insulin resistance are commonly associated with hyperlipidemia and high caloric fat- enricheddiets are widely accepted to induce hyperlipidemia [26].
A western-type diet (TD.88137) was chosen for thesestudies for its ability to induce hypercholesterolemia,
obesity and atherosclerosis depending on the genetic
background of the mice used for study [24, 27, 28]. Previous studies have shown that 16 weeks of WD regime issufficient to induce severe hypercholesterolemia in mice
[29, 30]. Augmentation of whole-body fat mass andmarkedly elevated circulatory cholesterol (especiallymales) was observed in Stap1+/+ and Stap1−/− mice atthe end of 16 weeks WD (Figs. 3 and 4). However, thesechanges were not exacerbated, as hypothesized, in micelacking global Stap1 expression and suggest the loss of
Stap1 does not exacerbate hyperlipidemia.
The link between STAP1 and hypercholesterolemia
was proposed when some FH4 patients were reported tohave variants of the gene, in the absence of changes inknown hyperlipidemia loci [6]. A review of genetic variants associated with FH noted 2580 in LDLR, 896 inKanuri et al. BMC Medical Genetics(2020) 21:234

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Fig. 3 Effect of western diet on growth parameters and body composition in mice lacking Stap1 gene. Panel a shows the growth curves in wildtype and knockout mice and no statistical differences were discernable in the patterns of weight gain on the high caloric diet (n = 6). Neither thepercent fat or lean mass were altered in Stap1−/− mice compared to age and sex-matched wildtype mice on a western diet (panel b, n = 6). Nochanges in weights of liver, spleen or kidneys were seen in male (panel c, n = 4–6) or female mice (panel d, n = 5–6) were noted. No changes inglucose tolerance tests were noted between knockout and wildtype controls either (panel e, n = 6). In case of male Stap1−/− mice, kidney weights
of only 2 mice were considered for analysis

APOB, 351 in PCSK9, 16 in APOE, and 4 in STAP1 [8].
Fouchier et al in 2014 demonstrated four variants in the
STAP1 gene of FH4 patients and characterized thephenotype. All four STAP1 variants were missense mutations (Glu97Asp, Leu69Ser, Ile71Thr, and Asp207Asn).
Together with the fact that the phenotype of STAP1
variant carriers was milder than other forms of ADH [6],one possibility is whether these are ‘false positive’ hits.
Nevertheless the association between STAP1 and ADH
was supported further by other studies showing elevatedcirculatory cholesterol in patients with STAP1 variants[10–12, 31]. However, this link has been highly debatedwith studies reporting no association between STAP1

and ADH [13–17], or no difference in plasma lipid profiles in carriers of STAP1 variants and controls [18].
Moreover, Loaiza et al recently also found the absenceof any significant difference in lipid phenotype in
Stap1−/− mice compared to controls, even after metabolic challenges [18]. The current study is a verificationof these findings. Nevertheless, a KO mouse model is aloss of function model and does not address the possibility of a gain of function variants in Stap1. The fact thatthe previously identified STAP1 variants were missense
mutations suggests that a viable protein may potentiallybe expressed in those patients, though its functionality
would be purely speculative.

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Fig. 4 Effect of 16 weeks of western diet (WD) on plasma lipid profiles. Panel a shows the plasma cholesterol and panel b the triglyceride levels inwildtype and knockout mice (n = 6 per group). Female mice (whether wild-type or knockout) seem to be resistant to a WD-induced
hypercholesterolemia (panel a), but this may be a feature of the mouse strain background. FPLC cholesterol profiles of the plasma (panel c, males,
panel d, females) showed that the diet in males led to a dyslipidemic profile, but loss of Stap1 did not affect these patterns. Error bars denote ±1 SD

It was initially hypothesized that extra-hepatic mechanisms were responsible for STAP1 mediated hypercholesterolemia due to the fact that liver distribution was
low [6, 31]. Interestingly, recent experiments using single
cell RNA-Seq indicated that its expression is highly specific to lymphoid cells (https://tabula-muris.ds.czbiohub.
org/) and any expression detected by examining tissue
RNA likely comes from the presence of these cells inthese tissues. Stap1 expression measured by qPCR in thecurrent study was low in liver and other tissues except
for the spleen (Fig. 1b). This could be explained by theconcentration of B-cells in the spleen and their presencein other organs, as the tissues were not perfused prior tocollection. Some studies have associated STAP1 variations with cardiovascular disease [10–12], and immune
cells have been linked with atherosclerosis and FH [32].
This study did not thoroughly investigate possible effects
of STAP1 deletion on the immune system, but there
were no obvious changes in B-cell subsets and IgG (datanot shown). Further studies would be obviously necessary to fully characterize the effects, if any, on immune
function. Nevertheless, transplant of Stap1−/− bonemarrow (BM) into Ldlr−/− female mice had no impact
on plasma lipids as well on the development of atherosclerosis plaques [18]. Furthermore, there were nomarked changes in B-lymphocyte populations in bloodof carriers of STAP1 gene variants compared to age- andsex-matched family controls [18].
The study by Loaiza et al was well-designed and thorough, but differs from the current investigation in two
important experimental variables: 1) the strategy used togenerate Stap1 KO mice, using clustered regularly interspaced short palindromic repeats/clustered regularlyinterspaced short palindromic repeat-associated 9 (CRIS
PR/Cas9) technology vs targeted homologous recombination of embryonic stem cells and 2) the much shortertime period for hyperlipidemia induction in global andtissue-specific KO mice (4 weeks vs 12 weeks). Since 16
weeks of WD regime is sufficient to induce severe
hypercholesterolemia and the fact that FH patients show
elevated levels of plasma cholesterol, the long-term regime that we used is ideally advantageous over the shortduration regime used in the study by Loaiza et al.
An important limitation of the study is loss-of-function
models do not recapitulate the missense variants reported
for STAP1 in humans. However, we are confident ourcurrent studies are valid as our characterization (in keeping with published data) finds no meaningful expressionof STAP1 in cells other than B-cells. Thus, even for missense variants of STAP1 to have a pathological role inKanuri et al. BMC Medical Genetics(2020) 21:234

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Fig. 5 Effect of western diet (WD) on liver morphology and lipids. Assessment of liver histology by hematoxylin and eosin staining showed afatty liver in all mice, irrespective of Stap1 status or sex (panel a and b). This was confirmed by analyses of liver cholesterol (panel c, n = 6 pergroup in males and 5 per group in females) and triglyceride (panel d, n = 6 per group in males and 5 per group in females) but loss of Stap1
does not affect these parameters. Error bars denote ±1 SD

causing hyperlipidemia in humans, a mechanistic pathway
to do so would need to be identified. Based upon ourcurrent understanding of lipid metabolism, no such meaningful pathway exists.

Conclusion
Global loss of Stap1 in mice does not result in an abnormal lipid phenotype, and this study validates the study
by Loaiza et al, who reported similar findings. Additionally, expression STAP1 mRNA seems to be confined to
B-cells, suggesting alteration of lipid metabolism in a
direct manner to be not feasible. Genetic changes in
STAP1 are therefore unlikely to be cause of familial
hypercholesterolemia and variants reported in such rarefamilies may represent a spurious association.
Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s12881-020-01176-x.
Additional file 1: Table S1. Primer sequences.
Additional file 2. Sequencing of off-target products on chromosome 2.
Additional file 3.

Abbreviations
ADH: Autosomal dominant familial hypercholesterolemia;
APOE: Apolipoprotein E; BRDG: B-cell antigen receptor downstream signaling
1 protein; FLPC: Fast Performance Liquid Chromatography; LDLR: Low
density lipoprotein receptor; OGTT: Oral glucose tolerance test;
PCR: Polymerase chain reaction; PCSK9: Proprotein convertase subtilisin/kexin
type 9; STAP1: Signal transducing adaptor family member protein 1;
WD: Western diet
Acknowledgments
We would like to acknowledge the technical assistance of Vicky Horton,
Casey Crane, Min Jiang, Ryan Ulrich, and William Thompson.
Authors’ contributions
SBP and DYH conceptualized, designed and supervised the study; AH
performed the experiments; BK and VF analyzed the data and drafted themanuscript. All authors read and approved the final manuscript.
Funding
This study was supported by institutional funds from the University ofCincinnati (SBP).
Availability of data and materials
The datasets used and analyzed during the current study are available fromthe corresponding author upon request. Requests for mice will require a
simple materials transfer agreement with the University of Cincinnati and all
costs for transfer will be the responsibility of the requestor.

Kanuri et al. BMC Medical Genetics(2020) 21:234

Ethics approval and consent to participate
There are no clinical studies involved in this work. All animal studies wereapproved by the Institutional Animal Care and Use Committee at the
University of Cincinnati (Protocol No. 16–04–19-01).
Consent for publication
All the authors have read and consented for publication of this article.

Page 9 of 9

16.

17.

Competing interests
The authors declare no competing interests.
18.
Author details
1
Division of Endocrinology, Diabetes and Metabolism, University ofCincinnati, Cincinnati, OH, USA. 2Department of Pathology, University ofCincinnati, Cincinnati, OH, USA.
19.
Received: 27 August 2020 Accepted: 15 November 2020

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