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\begin{document}

\title{Identification of a novel ANK1 c.856C\textgreater{}T nonsense mutation
in two patients from a Chinese family with hereditary spherocytosis by
NGS}



\author[1]{Jie Yang}%
\author[2]{Chun-xue Yang}%
\author[1]{Yuan-yuan Wu}%
\author[1]{Deng He}%
\author[1]{Yu-qi Li}%
\author[1]{Ling Feng}%
\author[1]{Shao-shuai Wang}%
\author[1]{zhi-hui Rong}%
\author[1]{Hai-Yi Liu}%
\affil[1]{Huazhong University of Science and Technology}%
\affil[2]{Wuhan No. 1 Hospital}%


\vspace{-1em}



  
  \date{May 22, 2024}


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\selectlanguage{english}
\begin{abstract}
Hereditary spherocytosis (HS) is a common inherited heterogeneous
hemolytic anemia that is characterized by the presence of spheroidal
erythrocytes on the peripheral blood smear. Mutations in ankyrin gene
(ANK1) is the most common cause of HS in Northern European populations
and Chinese patients but is seen in only 5--10\% of Japanese patients.
The majority of them are familial mutations inherited in an autosomal
dominant form. In this study, a heterozygous ANK1 c.856C\textgreater{}T
mutation was identified in a 2-hour-old newborn with severe jaundice
using targeted next-generation sequencing (NGS) and Sanger sequencing,
and was confirmed to be inherited from his mother.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Hereditary spherocytosis is a common inherited disorder that is
characterized by the presence of spheroidal erythrocytes on the
peripheral blood smear. Its prevalence ranging from 1:2,000 to 1:5,000
in north European and 1:100,000 in Chinese individuals {[}1, 2{]}. The
clinical manifestations of HS are variable, ranging from an asymptomatic
condition to severe hemolysis. Typical HS patients present anemia,
jaundice, splenomegaly and reticulocytosis {[}3{]}. In the neonatal
period, the major clinical presents are jaundice and anemia.
Splenomegaly and spherocytes are rarely observed {[}4{]}. Previous
researches have shown that mutations of five genes that encode the
erythrocyte membrane proteins are associated with HS, including ANK1,
SPTB, SPTA1, SLC4A1, and EPB42, which result in a loss of membrane
deformability and lead to surface area loss and an increased number of
peripheral blood spherocytes {[}1{]}. Approximately 75\% of HS cases are
inherited in an autosomal dominant manner and a subset of patients show
autosomal recessive inheritance or may carry a de novo mutation {[}5{]}.
Blood transfusion and splenectomy are the main treatment to correct
anemia and reduce clinical symptoms for HS patients.

The ANK1 gene is located at chromosome 8p11.2 and encodes several
alternative splices. ANK1 mutations have been implicated in
approximately half of all patients with hereditary spherocytosis (HS)
{[}6{]}, causing both dominant and recessive disease that can range from
clinically mild and severe. ANK1 contains 42 exons that encode an 1,881
amino acid protein (referred to NM\_000037.4), with three distinct
domains: a N-terminal domain containing multiple ankyrin repeats; a
center region with a spectrin-binding domain, and a C-terminal
regulatory domain {[}7{]}. By linking \selectlanguage{greek}β \selectlanguage{english}spectrin to band 3, ANK1 protein
leads to spectrin assemble on the membrane and stabilize the blood cell
membrane. NGS provides a comprehensive and cost-effective approach to
molecular diagnosis of hereditary hemolytic anemia, including HS
{[}8{]}.

In this study, a heterozygous ANK1 c.856C\textgreater{}T mutation was
identified by NGS and Sanger sequencing in a 2-hour-old newborn and his
mother whom had been suffered anemia, jaundice and splenomegaly during
her young time and received a splenectomy at the age of 12.

\textbf{Results} .

\textbf{Clinical history}

The affected male newborn presented with jaundice without other
pathological symptoms or signs since 2 hours after birth. Then, the
routine blood examination showed that he suffered from mild hemolytic
anemia with neonatal hyperbilirubinemia and sphere-shaped erythrocytes
on peripheral blood smear (Figure 1B). And his Hb values decreased
sharply over the subsequent 9 days (Table.1). The erythrocyte osmotic
fragility test was positive while both glucose-6-phosphate
dehydrogenase(G-6-PD) screening test and Coombs' test were negative.
Hemoglobin electrophoresis analysis using for thalassemia preliminary
screen was negative. High bilirubin susceptibility gene (5 mutation
sites of UGT1A1) mutation test using Sanger sequencing were also
negative. The male newborn took treatment in hospital for 2 weeks,
including twice blood transfusions.

Therefore, we traced the family history again and found that the mother
had undergone a total splenectomy for severe hemolysis and jaundice when
she was 12 years-old. The proband's farther and sister were clinically
and hematologically normal. This potentially suggested a dominant
inheritance of disease phenotype from mother to the proband. This was
another HS indicator, as HS patients with severe symptoms often get
better after a splenectomy. The family tree is shown in Figure 1A, and
the laboratory tests are summarized in Table 1.

\textbf{Identification of ANK1 mutation}

A heterozygous ANK1 c.856C\textgreater{}T mutation was identified using
NGS. Sanger sequencing of the ANK1 c.856C\textgreater{}T mutation was
performed on all family members. The mutation is a substitution of
C\textgreater{}T, at the 46 nucleotide in exon 9. Both the patient and
his mother were heterozygous for the ANK1 c.856C\textgreater{}T
mutation, while his father had a wild-type ANK1 allele. The genetic
analysis for his sister was not available (Figure 1C). This mutation was
not found in the gnomAD, 1000G, ExAC and HGMD database, and confirmed as
a novel mutation. The mutation was a heterozygous mutation.

\textbf{Computational analysis}

We used Swiss-model to visualize 3-D representations of the protein. The
variation caused a substitution from Arginine acid to a premature stop
at codon 286 and get a truncated ANK1 protein (Figure 2).

\textbf{Genetic and phenotype association analysis}

Genetic tests showed a heterozygous ANK1 c.856C\textgreater{}T mutation
in this family, and both patients carried this mutation. HS associated
clinic symptoms are observed in the patients. Consistent with this
genetic finding, the mother who received a splenectomy in childhood, is
now asymptomatic.

\textbf{Discussion}

ANK1 Mutation is the most common cause of HS. ANK1 mutations is
inherited in both AD (autosomal dominant) and AR (autosomal recessive)
patterns in HS and the majority is de novo mutation {[}1{]}. These
mutations locate the entire ANK1 gene, throughout the promoter region
and coding exons {[}12, 13{]}. To date, a total of 62 mutations are
included in the HGMD public database. Missense/nonsense and small
deletions have been identified in approximately 70\% of all mutations in
the ANK1 gene. Seven splicing mutations, occurring in IVS1,16,20,22,28
and 38, are included in the HGMD database. In this study, we described a
Chinese family with a proband and his mother affected by HS. A de novo
mutation (c.856C\textgreater{}T p.R286*) causing a premature stop codon
in exon 9 of ANK1 was found by investigating this family through NGS
followed by Sanger sequencing to certify the relationship between the
ANK1 mutation and HS.

ANK1 plays a pivotal role in the stabilization of the membrane,
providing the main membrane binding site for the spectrin-based membrane
skeleton. ANK1 consists of three structural domains: a multiple repeats
N-terminal domain(89kD), a spectrin-binding center region(62kD) and a
regulatory C-terminal domain(55kD) {[}7, 14, 15{]}. Mutations in the
spectrin-binding domain and regulatory C-terminal domains result in the
most severe anemia compared with those located in the other domains
{[}14, 16{]}. In this family, a ANK1 c.856C\textgreater{}T mutation
occurred in the N-terminus region. This mutation was not found in the
gnomAD ,1000G, ExAC and HGMD database, and confirmed as a novel
mutation. According to bioinformatics analyses, this point mutation
generates a premature translation termination codon resulting in a
truncated ANK1 protein, losing the important spectrin-binding and
regulatory C-terminal domains and leaving only partial of the N-domain,
which might cause HS. Moreover, consistent with the genetic diagnosis,
the symptom of the mother was ameliorated after splenectomy, further
supporting the genotype-phenotype relationship.

NGS provides a comprehensive and cost-effective approach to molecular
diagnosis of hereditary hemolytic anemia, especially in cases where
biochemical testing is unreliable due to multiple transfusions. In
previous research {[}17{]}, a female individual presented with a yellow
complexion, jaundice and splenomegaly without other pathological
symptoms or signs on the second day after birth. And then the girl who
was clinically diagnosed with HS and under laparoscopic splenectomy
because of splenomegaly and hemolysis when she was 6-year-old. Finally,
using NGS detected a de novo nonsense ANK1 mutation (c.796G
\textgreater{} T, p. Glu266X), which caused a substitution from glutamic
acid to a premature stop at codon 266 and may lead to HS. But no
mutation was detected in her parents. Thus, it is efficient and
important for us to using NGS to identify inherited, rare gene mutations
which are associated with a high risk for the development of diseases.
If diagnosed earlier, fewer newborn infants would develop severe anemia
and hazardous hyperbilirubinemia and it would remind pediatricians to
closely monitoring of infants with HS during the first 6 months of life
that is important for appropriate clinical management.

In summary, our results demonstrated an ANK1 c.856C\textgreater{}T
mutation may lead to a premature stop at codon 286 and may be
responsible for HS in two patients from a Chinese family. Identifying
the underlying genetic cause not only helps with the management of the
patients, it also facilitates accurate genetic counselling and helps in
guidance regarding the predicted severity of clinical phenotype.

\textbf{Reference}

1. Silverio Perrotta 1, Patrick G Gallagher, Narla Mohandas. Hereditary
spherocytosis. Lancet 2008,18;372(9647):1411-26.

2. Wang, C., Cui, Y., Li, Y., Liu, X., and Han, J. A systematic review
of hereditary spherocytosis reported in Chinese biomedical journals from
1978 to 2013 and estimation of the prevalence of the disease using a
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3. Deng Z, Liao L, Yang W, Lin F: Misdiagnosis of two cases of
hereditary spherocytosis in a family and review of published reports.
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4. Christensen RD, Yaish HM, Gallagher PG: A pediatrician's practical
guide to diagnosing and treating hereditary spherocytosis in neonates.
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5. Lux, S.E., and Palek, J. Disorders of the red cell membrane. In:
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6. Eber SW, Gonzalev JM, Lux ML, et al. Ankyrin-1 mutations are a major
cause of dominant and recessive hereditary spherocytosis. Nat Genet.
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7. Gallagher PG. Hematologically important mutations: ankyrin variants
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10. Richards S, Aziz N, Bale S, et al. Standards and Guidelines for the
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13. H Nakanishi 1, A Kanzaki, A Yawata, O Yamada, Y Yawata. Ankyrin gene
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\textbf{Figure legend}

Figure 1. Identification of a novel ANK1 c.856C\textgreater{}T nonsense
mutation in two patients from a Chinese family with hereditary
spherocytosis. (A)Pedigree of Chinese family with HS. Squares indicate
male and circles indicate female. Black symbols denote patients with HS.
A black arrow indicates the Proband. (B)Peripheral blood smear. A black
arrow indicates spherical-shaped erythrocytes. (C)Sanger sequencing
identified an ANK1 c.856C\textgreater{}T mutation in the patients. A red
arrow indicates the mutation site.

Figure 2. Protein spatial structure analysis. (A) Intact protein
structure of ANK1. (B) Nonsense ANK1 mutation (c.856C\textgreater{}T,
p.R286*), caused a substitution from Arginine acid to a premature stop
at codon 286. A truncated ANK1 protein, losing the important
spectrin-binding and regulatory C-terminal domains and leaving only
partial of the N-domain.\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/Figure-1/Figure-1}

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\includegraphics[width=0.70\columnwidth]{figures/Figure-2/Figure-2}

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\textbf{Hosted file}

\verb`Table 1.docx`  available at \url{https://authorea.com/users/728523/articles/709566-identification-of-a-novel-ank1-c-856c-t-nonsense-mutation-in-two-patients-from-a-chinese-family-with-hereditary-spherocytosis-by-ngs}

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