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\begin{document}

\title{Phenotypic homozygous familial hypercholesterolemia successfully treated
with proprotein convertase subtilisin/kexin type 9 inhibitors}



\author[1]{Ryosuke Tani}%
\author[2]{Keiji Matsunaga}%
\author[3]{yuta toda}%
\author[2]{Tomoko Inoue}%
\author[1]{hai fu}%
\author[2]{Tetsuo Minamino}%
\affil[1]{Faculty of Medicine, Kagawa University, Cardiorenal and Cerebrovascular Medicine}%
\affil[2]{Faculty of Medicine, Kagawa University}%
\affil[3]{Kagawa University Hospital}%


\vspace{-1em}



  
  \date{June 14, 2024}


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\textbf{Manuscript type}

Case Report

\textbf{Title}

Phenotypic homozygous familial hypercholesterolemia successfully treated
with proprotein convertase subtilisin/kexin type 9 inhibitors

\textbf{Author information}

Ryosuke Tani, Keiji Matsunaga, Yuta Toda, Tomoko Inoue, Hai Ying Fu,
Tetsuo Minamino

Department of Cardiorenal and Cerebrovascular Medicine, Faculty of
Medicine, Kagawa University, Kagawa, Japan.

Correspondence to: Keiji Matsunaga M.D, Ph.D.

Department of Cardiorenal and Cerebrovascular Medicine, Faculty of
Medicine, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa
Prefecture 761-0793, Japan.

Tel: +81-87-891-2150; Fax: +81-87-891-2151

E-mail:\emph{matsunaga.keiji@kagawa-u.ac.jp}

\textbf{DATA AVAILABILITY STATEMENT}

All data generated or analysed during this study are available as part
of the article, and no additional data sources are required.

\textbf{FUNDING STATEMENT}

None.

\textbf{CONFLICT OF INTEREST DISCLOSURE}

None to declare.

\textbf{ETHICS APPROVAL STATEMENT}

This study was conducted in accordance with the declaration of Helsinki.

\textbf{PATIENT CONSENT STATEMENT}

Written informed consent was obtained from the patient for publication
in accordance with the journal's patient consent policy.

\textbf{Key Clinical Message}

Recent data reveal phenotypic HoFH patients may be responsive to PCSK9
inhibitors, challenging prior assumptions. Genetic testing advancements
now more accurately forecast patient responses to these therapies,
improving treatment strategies.

\textbf{Keywords}

homozygous familial hypercholesterolemia, pediatric familial
hypercholesterolemia, proprotein convertase subtilisin/kexin type 9
inhibitors, genetic testing

\textbf{Main text}

\textbf{INTRODUCTION}

Familial hypercholesterolemia (FH) is a genetic lipoprotein disorder
characterised by elevated plasma low-density lipoprotein cholesterol
(LDL-C) levels and early onset of atherosclerotic premature coronary
artery disease (PCAD).\textsuperscript{1-3} This condition manifests
distinct clinical features such as xanthomas, thickening of the Achilles
tendon, and corneal rings, which often serve as clinical pointers even
before genetic confirmation. In particular, homozygous familial
hypercholesterolemia (HoFH) is a severe form, presenting with xanthomas
and supravalvular aortic stenosis from early childhood and requiring
different management from heterozygous familial hypercholesterolemia
(HeFH).\textsuperscript{4,5}

In this case, cutaneous xanthomas before the age of 10 years, thickening
of the Achilles tendon, and high levels of LDL-C were suspected to be
homozygous HoFH. Historically, our understanding of FH, especially HoFH,
suggested that this form was generally unresponsive to proprotein
convertase subtilisin/kexin type 9 (PCSK9) inhibitors, primarily because
of the absence of low-density lipoprotein (LDL) receptor
activity.\textsuperscript{6} This viewpoint has significantly influenced
pharmacotherapeutic decisions, often limiting the available treatment
options for patients. This paper reports a case of phenotypic HoFH that
was successfully treated with PCSK9 inhibitors.

\textbf{CASE REPORT}

\textbf{Case history/Examination}

A 29-year-old woman presented with cutaneous xanthomas before the age of
10 years and had undergone multiple resections of xanthomas in her
teens. The patient was referred to our hospital when xanthomas
reappeared. She had Achilles tendon thickening, nodular xanthoma on the
elbows, and palpebral xanthoma. No palmar xanthomas or corneal rings
were observed.

She had a smoking history of 10 cigarettes per day since the age of 20
years with minimal alcohol consumption. In terms of her family history,
her mother suddenly died at the age of 60 years. While the exact cause
remains unknown, the circumstances could not rule out the possibility of
PCAD. Additionally, her sister had undergone multiple resections for
xanthomas during childhood.

On examination, her temperature was 36.0; her blood pressure was 108/68
mm Hg; her pulse was 60/min; and her oxygen saturation was 98\% on
ambient air. Blood tests revealed total cholesterol (TC) of 429 mg/dL,
low-density lipoprotein cholesterol level of 357 mg/dL, high-density
lipoprotein cholesterol (HDL-C) of 45 mg/dL, triglyceride (TG) of 136
mg/dL, and lipoprotein (a) of 20.2 mg/dL. The Achilles tendon showed a
thickening of 15 mm on radiography.

\textbf{Differential diagnosis and investigations}

In this patient, cutaneous xanthomas before the age of 10 years,
thickening of the Achilles tendon, and a high LDL-C were suggestive of
phenotypic HoFH. We also considered other genetic lipid disorders, such
as sitosterolemia, lysosomal acid lipase deficiency, and
cerebrotendinous xanthomatosis. However, the patient exhibited normal
sitosterol and lysosomal acid lipase activities. In addition, the
patient showed no neurological symptoms typically associated with
cerebrotendinous xanthomatosis.

Following a comprehensive discussion with the patient about the risks,
benefits, and implications of genetic testing, informed consent was
obtained, and genetic testing was performed. Genetic testing by
next-generation sequencing (NGS) through exome analysis targets 22 genes
associated with lipid metabolism abnormalities including low-density
lipoprotein receptor (\emph{LDLR} ), apolipoprotein B (\emph{APOB} ),
proprotein convertase subtilisin/kexin type 9 (\emph{PCSK9} ),
low-density lipoprotein receptor adaptor protein1(LDLRAP1), ATP binding
cassette subfamily G member 5 (\emph{ABCG5} ), ATP binding cassette
subfamily G member 8 (\emph{ABCG8} ), apolipoprotein E (\emph{APOE} ),
and cytochrome P450 family 27 subfamily A member 1 (\emph{CYP27A1} )
detected no pathogenic or likely pathogenic variant. In addition,
multiple ligation-dependent probe amplifications (MLPA) of \emph{LDLR}
revealed a large deletion in exons 7-14.

\textbf{Outcome, treatment, and follow-up}

Based on these results, the patient was diagnosed with phenotypic HoFH.
Initial therapy with maximum tolerated doses of rosuvastatin and
ezetimibe reduced the LDL-C levels to 152 mg/dL. However, a target LDL-C
level of \textless{}55 mg/dL was not achieved. Treatment with a PCSK9
inhibitor (evolocumab) was initiated, considering the residual LDL
receptor activity. This intervention proved highly effective, decreasing
the patient's LDL-C level to 22 mg/dL (Table 1). No medication side
effects were observed, adequate LDL-C control was achieved, and the
palpebral xanthoma regressed. Written informed consent was obtained from
the patient for publication in accordance with the journal's patient
consent policy.

\textbf{DISCUSSION}

There are two primary classifications of FH: HeFH and HoFH. Individuals
with HeFH possess one pathogenic variant and show an abnormal
biochemical phenotype. In contrast, those with genotypic HoFH have two
variants, which lead to more severe LDL-C deviations and symptoms. The
primary genes associated with these conditions include \emph{LDLR}
,\emph{APOB} , \emph{PCSK9} , and \emph{LDLRAP1} .\textsuperscript{7} To
ensure clarity and avoid misinterpretation, the recent guidelines
recommend using the term ``phenotypic HoFH'' as a concise clinical
descriptor when a genetic diagnosis is not confirmed.\textsuperscript{8}

This case involved a patient who was initially considered to have
phenotypic HoFH. The patient responded well to PCSK9 inhibitors, and
detailed genetic testing confirmed the diagnosis of HeFH. This case
study highlights two important aspects. Some cases of phenotypic HoFH
are notably treated with LDL receptor-mediated pharmacotherapy,
including PCSK9 inhibitors. Second, in cases of phenotypic HoFH,
detailed genetic testing may help predict the responses to these
pharmacotherapies (Figure 1).

HoFH is known to be ineffective with LDL receptor-mediated
pharmacotherapy, and LDL apheresis might be considered a treatment
option.\textsuperscript{9} HoFH typically exhibits a near-complete
absence of LDL receptor activity, making it less responsive to
pharmacotherapy, such as statins and PCSK9 inhibitors, by upregulating
LDL receptors. Conversely, patients with HeFH retain some LDL receptor
activity and may benefit significantly from these pharmacotherapies. The
term ``phenotypic HoFH'' generally encompasses not only true homozygotes
but also double heterozygotes and compound
heterozygotes.\textsuperscript{10} However, the patient exhibited
phenotypic HoFH, but genetic testing indicated that she was heterozygote
with large-scale copy number variations (CNVs) deletion. In this case,
it was revealed that this type also exhibited phenotypic HoFH.
Furthermore, HoFH in true homozygotes typically exhibits a near-complete
absence of LDL receptor activity; however, there is potential for
residual LDL receptor activity in heterozygotes. Therefore, these
individuals may benefit from LDL receptor-mediated pharmacotherapy. In
this case, the patient exhibited phenotypic HoFH but was suspected to
retain some LDL receptor activity. This residual activity likely
contributes to the notable effectiveness of the PCSK9 inhibitor.

Further, in cases of phenotypic HoFH, detailed genetic testing may help
predict the responses to these pharmacotherapies. The detection rate of
pathogenic variants in clinically diagnosed cases of FH is reportedly
approximately 60--80\%.\textsuperscript{11,12} Genetic testing for FH is
not mandatory; it can make the diagnosis of FH more definitive, and if
the originator has been genetically tested, the diagnosis of FH in the
family is also assured.\textsuperscript{13,14} There are several methods
of genetic testing. NGS is renowned for its ability to sequence millions
to billions of DNA strands simultaneously.\textsuperscript{15} This
allows for a broad examination of substantial genomic regions in a
single run. However, implementing this approach is challenging. While
NGS can potentially detect variations, such as single nucleotide
variants, insertions, and deletions, it is sometimes less reliable for
detecting CNVs.

In contrast, MLPA is used to detect large-scale deletions or
amplifications of genes that are difficult to analyse using whole-gene
sequencing methods. The data derived from MLPA are quantitative,
rendering an accurate representation of CNVs compared to
NGS.\textsuperscript{16} However, they are limited in scope and are
incapable of detecting single-nucleotide variants (SNVs). Most of the
pathogenic variants of FH cases are SNVs in \emph{LDLR} , but
approximately 10\% are attributed to CNVs in\emph{LDLR}
.\textsuperscript{17} Therefore, a single genetic test on one side alone
may not detect the genetic mutation or may be misjudged. Detailed
genetic testing combining multiple genetic tests may provide an
efficient algorithm for the genetic testing of FH\textsuperscript{18}.
It may be useful in assessing residual LDL receptor activity and
predicting the response to these pharmacotherapies in cases exhibiting
phenotypic HoFH. The point of our case lies in the phenotypic HoFH
patient who demonstrated an unexpectedly positive response to PCSK9
inhibitors, emphasising the need for a deeper dive into individual
genetic phenotypes.

\textbf{Limitation}

Some cases of phenotypic HoFH are notably responsive to LDL
receptor-mediated pharmacotherapy, including PCSK9 inhibitors, and
detailed genetic testing may help predict the response to these
pharmacotherapies. However, further studies are needed to confirm these
hypotheses. For cases where PCSK9 inhibitors are ineffective, recent
developments in angiopoietin-like protein 3 (ANGPTL3) inhibitors, which
have shown efficacy even in HoFH cases without LDL receptor activity,
offer a promising avenue.\textsuperscript{19} ANGPTL3 inhibitors offer
the potential to attain the LDL-C goal or reduce the need for LDL
apheresis and show promising potential for determining optimal
pharmacotherapies in the future.

\textbf{Conclusion}

While HoFH is traditionally considered ineffective with LDL
receptor-mediated pharmacotherapy, there are phenotypic HoFH cases that
demonstrate notable effectiveness with treatments such as PCSK9
inhibitors. Detailed genetic testing may help predict responses to these
pharmacotherapies.

\textbf{Figure 1}

HoFH, homozygous familial hypercholesterolemia; LDL, low-density
lipoprotein; CNVs, copy number variations; PCSK9, proprotein convertase
subtilisin/kexin type 9; ANGPTL3, angiopoietin-like protein 3.

\textbf{Table 1}

Lipid profile of the patient.

\textbf{Author Contributions}

Ryosuke Tani: Conceptualisation, data curation, investigation,
resources, and writing the original draft.

Keiji Matsunaga: Supervision; writing review and editing.

Yuta Toda: Data curation; investigation.

Tomoko Inoue: Data curation; investigation.

Hai Ying Fu: Data curation; investigation.

Tetsuo Minamino: Supervision; writing review and editing.

\textbf{ACKNOWLEDGMENTS}

We thank Akane Yabuchi and Tomoko Nakayama for their assistance with
patient data collection.

\textbf{References}

1. Frederick J. Raal, G. Kees Hovingh, Alberico L. Catapano. Familial
Hypercholesterolemia Treatments: Guidelines and New
Therapies.\emph{Atherosclerosis} . 2018; 277:483-492.

2. Mariko Harada-Shiba, Hidenori Arai, Hirotoshi Ohmura, et al.
Guidelines for the diagnosis and treatment of adult familial
hypercholesterolemia 2022. \emph{J Atheroscler Thromb} .
2023;30(5):558-586.

3. Mariko Harada-Shiba, Akira Ohtake, Daisuke Sugiyama, et al.
Guidelines for the Diagnosis and Treatment of Pediatric Familial
Hypercholesterolemia 2022. \emph{J Atheroscler Thromb} .
2023;30:531-557.

4. Frederick J. Raal, Raul D. Santos. Homozygous familial
hypercholesterolemia: Current perspectives on diagnosis and
treatment.\emph{Atherosclerosis} . 2012;223(2):262-268.

5. Mary P. McGowan, Seyed Hamed Hosseini Dehkordi, Patrick M. Moriarty,
et al. Diagnosis and treatment of heterozygous familial
hypercholesterolemia. \emph{Journal of the American Heart Association} .
2019;8(24):e013225.

6. B\selectlanguage{ngerman}élanger AM, Akioyamen L, Alothman L, et al. Evidence for improved
survival with treatment of homozygous familial
hypercholesterolemia.\emph{Curr Opin Lipidol} . 2020;31(4):176-181.

7. Berberich AJ, Hegele RA. The complex molecular genetics of familial
hypercholesterolemia. \emph{Nat Rev Cardiol} . 2019;16(1):9-20.

8. Marina Cuchel, Frederick J. Raal, Robert A. Hegele, et al. 2023
Update on European Atherosclerosis Society Consensus Statement on
Homozygous Familial Hypercholesterolemia: New treatments and clinical
guidance. \emph{Eur Heart J} . 2023;44(25):2277-2291.

9. Christina Taylan, Lutz T. Weber. An update on lipid apheresis for
familial hypercholesterolemia. \emph{Pediatr Nephrol} .
2023;38(2):371-382.

10. Frederick J. Raal, Barbara Sjouke, G. Kees Hovingh, et al. Phenotype
Diversity among Patients with Homozygous Familial Hypercholesterolemia:
A Cohort Study. \emph{Atherosclerosis} . 2016;248:238-244.

11. Hiroshi Mabuchia, Atsushi Nohara, Tohru Noguchi, et al. Molecular
genetic epidemiology of homozygous familial hypercholesterolemia in the
Hokuriku district of Japan. \emph{Atherosclerosis} .
2011;214(2):404-407.

12. Naotaka Ohta, Mika Hori, Atsushi Takahashi, et al. Proprotein
Convertase Subtilisin/Kexin 9 V4I Variant with LDLR Mutations Modifies
the Phenotype of Familial Hypercholesterolemia. \emph{Journal of
Clinical Lipidology} . 2016;10(3):547-555.

13. Emily E. Brown, Amy C. Sturm, Marina Cuchel, et al. Genetic Testing
in Dyslipidemia: A Scientific Statement from the National Lipid
Association. \emph{Journal of Clinical Lipidology} . 2020;14:398--413.

14. Ahsen Chaudhry, Mark Trinder, Kristin Vesely, et al. Genetic
Identification of Homozygous Familial Hypercholesterolemia by Long-Read
Sequencing among Patients with Clinically Diagnosed Heterozygous
Familial Hypercholesterolemia. \emph{Circulation: Genomic and Precision
Medicine} . 2023;16(2):e003887.

15. Laurens F. Reeskamp, Tycho R. Tromp, Joep C. Defesche, et al.
Next-Generation Sequencing to Confirm Clinical Familial
Hypercholesterolemia. \emph{European journal of preventive cardiology} .
2021;28(8):875-883.

16. Jian Wang, Matthew R. Ban, Robert A. Hegele. Multiplex
ligation-dependent probe amplification of LDLR enhances molecular
diagnosis of familial hypercholesterolemia. \emph{J Lipid Res} .
2005;46(2):366-372.

17. Michael A. Iacocca, Jian Wang, Jacqueline S. Dron, et al. Use of
Next-Generation Sequencing to Detect LDLR Gene Copy Number Variation in
Familial Hypercholesterolemia. \emph{Journal of lipid research} .
2017;58(11):2202-2209.

18. Chin-Chou Huang, Dau-Ming Niu, Min-Ji Charng. Genetic analysis in a
Taiwanese cohort of 750 index patients with clinically diagnosed
familial hypercholesterolemia. \emph{J Atheroscler Thromb} .
2022;29(5):639-653.

19. Frederick J. Raal, Robert S. Rosenson, Laurens F. Reeskamp, et al.
Evinacumab for homozygous familial hypercholesterolemia. \emph{N Engl J
Med} . 2020;383(8):711-720.\selectlanguage{english}
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