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\begin{document}

\title{Clinical and genetic characteristics in maternally inherited diabetes
and deafness (MIDD) with mitochondrial m.3243A\textgreater{}G mutation:
A 10-year follow up observation study of 1 family}



\author[1]{Shasha Zheng}%
\author[2]{Juanjuan Wang}%
\author[2]{Minxian Sun}%
\author[2]{Pei Wang}%
\author[3]{Wei Shi}%
\author[3]{Zhongzhi Zhang}%
\author[2]{Zhongjing Wang}%
\author[2]{Hongmei Zhang}%
\affil[1]{Central Hospital of Wuhan}%
\affil[2]{Huazhong University of Science and Technology Tongji Medical College Affiliated Wuhan Central Hospital}%
\affil[3]{Hubei Provincial Hospital of Integrated Chinese and Western Medicine}%


\vspace{-1em}



  
  \date{May 24, 2024}


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\textbf{Introduction}

Mitochondrial disease (MD) is a group of disorders caused by
dysfunctional mitochondria, the organelles which play an important role
in the production of ATP and exist in every human cell inthe body,
except for the red blood cells (\emph{1} ). Mitochondrial DNA (mtDNA) is
inherited maternally and affects organs dependent on high aerobic
metabolism, such as the eye, inner ear, central nervous system, skeletal
and cardiac muscle. Mitochondrial diabetes mellitus (MDM), also known
internationally as maternally inherited diabetes and deafness (MIDD)
syndrome, is a mtDNA mutation disease, with a progressive islet \selectlanguage{greek}β \selectlanguage{english}cell
secretory dysfunction (\emph{2, 3} ). MIDD is associated with early
onset diabetes and sensorineural deafness, but there are various other
systemic features, including cardiomyopathy, renal problems, and
neuropsychiatric symptoms. It was first reported by J.A. Massen et al.
in 1992, who found a large pedigree with mt.3243A\textgreater{}G
mutation, suffering from diabetes with the presence of maternal
transmission, in conjunction with bilateral hearing loss in most of the
carriers (\emph{4} ). From then on, there has been growing scientific
evidence that a range of other point mutations in mtDNA could contribute
to the pathogenesis of MIDD, such as mt-tRNA encoding genes, including
MT-TI, MT-TS1, and MT-TK, and mt-proteins encoding genes, incuding
MT-ND1, MT-ND4, MT-COX2, and MT-COX3. In addition, deletion and
depletion of nucleotides have also been described in patients with MIDD
(\emph{5} ). These novel mutations, however, are extremely rare compared
to the proportion of mt.3243A\textgreater{}G, where there is an A to G
substitution at position 3243. MIDD is currently found to be the most
common type of monogenic diabetes mellitus, accounting for about 1\% of
all diabetes mellitus, and the incidence in China is approximately 0.6\%
(\emph{6} ). Because of its low incidence and irrespective of its
complex clinical phenotype, MIDD is often misdiagnosed as type 1 or type
2 diabetes. Nonetheless, its treatment is different from the common
types of diabetes, and incorrect diagnosis and treatment will accelerate
the disease process and the occurrence of complications. Therefore,
correct genetic diagnosis and treatment are crucial for patients with
MIDD. This study discusses the clinical manifestations and treatment
process of a typical patient with MIDD, and summarizes its clinical
characteristics through a comprehensive review of the literature, with
the scope of improving clinicians' cognition of the disease and reducing
misdiagnosis rate.

\textbf{Materials and methods}

\textbf{Participants}

Age of at diagnosis was estimated by the patient's recall, as well as
the review of medical records and a detailed medical and family history
was obtained. We collected this large family of four generations,
ranging in age from 20 to 80 years, including 15 females and 9 males.
Classification and diagnosis of diabetes were in accordance with ADA's
latest guidelines (\emph{7} ). This study was approved by the ethics
committee of the Central Hospital of Wuhan in China. Peripheral blood
was collected from the proband, his mother and two daughters. Written
informed consent was obtained from all participants.

\textbf{Sample preparation and whole mitochondrial DNA sequencing}

Total DNA was extracted from the peripheral blood samples of the
participants (the proband, his mother and his both daughters) using a
TIANGEN DNA extraction kit (TIANGEN, Beijing, China). Sample preparation
and pretreatment for next generation sequencing were performed using the
SureSelect XT Low Input Reagent Kit Agilent (G9703A). MtDNA sequencing
was carried out on an HiSeq2500 system (Illumina), including MT-DLOOP,
MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-ND4L, MT-ND5, MT-ND6, MT-CYB, MT-CO1,
MT-CO2, MT-CO3, MT-ATP6, MT-ATP8, MT-TA, MT-TR, MT-TN, MT-TD, MT-TC,
MT-TE, MT-TQ, MT-TG, MT-TH, MT-TI, MT-TL1, MT-TL2, MT-TK, MT-TM, MT-TF,
MT-TP, MT-S1, MT-TS2, MT-TT, MT-TW, MT-TY, MT-TV, MT-RNR1 and MT-RNR2.
The resulting sequences were compared with the updated Cambridge
sequence (GenBank accession number: NC\_012920). Blast homology searches
were performed using the programs available at the National Center for
Biotechnology Information website and were compared with the wild-type
sequence. Areas containing putative novel variations were amplified and
sequenced again on both strands to exclude possible polymerase chain
reaction (PCR) artifacts.

\textbf{Sanger sequencing}

The presence of candidate variants in the proband and his family was
confirmed by direct Sanger sequencing of the PCR products, after which
the variants co-segregated with with diabetes were selected for further
analysis. The presence of mt.3243A\textgreater{}G variant in the proband
and his family(mother) was also confirmed by direct Sanger sequencing of
the PCR products. The MT-TL1 gene was amplified in a PCR assay using the
forward primer 5'-CCGGAGTAATCCAGGTCGGT-3' and thereverse primer
5'-CAGCATTCCCCCTCAAACCT-3'. The PCR assay conditions were as follows: 95
\selectlanguage{ngerman}°C for 5 min; 35 cycles, at 95 °C for 1 min, 60 °C for 30 sec, and 72 °C
for 1 min; and then 72 °C for 10min. The PCR products were assessed by
direct Sanger sequencing (Xiangyin Medical Testing Center, Hangzhou,
China).

\textbf{Medication plan adjustments}

Patients' treatment plans were adjusted based on genetic testing results
and previously reported findings. For example, insulin should be
administered to the proband, and melbine should never be used.

\textbf{Results}

\textbf{Medical history}

The proband was a 33-year-old man, who was hospitalized for symptoms
such as poor glycemic control and deafness for 11 years and abdominal
pain and vomiting for 1 day. The patient had a diagnosis of type 1
diabetes at the age of 22 years. During the clinical course of the
disease, the patient repeatedly experienced dry mouth, polydipsia,
polyuria, fatigue, weight loss, limb convulsions, abdominal pain,
vomiting, and repeated hospitalizations for ketosis. His previous
hospital records did not indicate conventionally-assessed islet
autoantibodies, and oral hypoglycemic drugs were found to be
ineffective. Figure 1 shows a pedigree chart of the proband's family.
His family history indicated that his mother had diabetes and his father
was a healthy individual without any family history of diabetes. His
grandmother (I-2), grandaunt (I-3), granduncle (I-4), mother (II-1),
five aunts (II-2, II-3, II-4, II-5, and II-8), one uncle (II-6), and one
cousin (III-5) experienced similar diseases, but the offspring of the
granduncle (II-9, II-10, II-11, and II-12), his grandfather (I-1), one
uncle (II-7), and five cousins (III-2, III-3, III-4, and III-6) did not
have diabetes. The proband's family had more women than men, and the
offspring of the men did not have diabetes. These findings conclude that
an extensive history of early-onset adult diabetes exsits within the
maternal lineage.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1.} Pedigree chart of the patient's family. Generations
are indicated on the left in Roman numerals, and the numbers under the
individuals represent identification numbers for each generation.
Squares represent males, and circles represent females. Black arrow
indicates the proband (III-1). Individuals with diabetes are indicated
as black symbols, and oblique line indicates death. The proband (III-1)
had a diagnosis of type 1 diabetes mellitus at the age of 22 years, and
his grandmother (I-2), grandaunt (I-3), granduncle (I-4), mother (II-1),
five aunts (II-2, II-3, II-4, II-5, and II-8), one uncle (II-6), and one
cousin (III-5) were diagnosed with type 2 diabetes mellitus at the age
of 40, 40, 45, 50, 50, 30, 40, 40, 50, 40, and 28 years, respectively.

\textbf{Clinical characteristics}

The clinical characteristics of the proband's family are presented in
Table 1 and Table 2. A total of five medical records of the proband were
collected during 2013-2022. The proband's laboratory analysis results
revealed poor glycemic control, and he was always hospitalized for
diabetic ketosis or ketoacidosis. The patient was lean, with a weight of
52 kg and a height of 1.70 m with a body mass index (BMI) of only 17.99
kg/m\textsuperscript{2}, in 2022. His fasting and 2-h C-peptide levels
were 0.6 and 3.0 ng/mL, respectively, in 2014, which decreased to 0.32
and 0.63 ng/mL, respectively, in 2022, indicating that the patient had
progressive islet \selectlanguage{greek}β-\selectlanguage{english}cell secretory dysfunction. He tested negative for
all diabetes autoantibodies in 2022. His blood lipid profile slightly
elevated or generally normal, but uric acid levels were high, possibly
because of ketoacidosis. In 2022, ultrasound examination of the liver,
kidney, pancreas, bladder, ureter, spleen, and gall bladder showed
normal organ morphology, but complications of the eye and lower
extremity artery showed faster progression. He was had a diagnosis of
glaucoma in 2013 and diagnosed with diabetic retinopathy in 2014 after
the diagnosis of glaucoma was ruled out. The patient reported as being
diagnosed with type 1 diabetes mellitus (T1DM) at the age of 22 years,
and his grandmother (I-2), grandaunt (I-3), granduncle (I-4), mother
(II-1), five aunts (II-2, II-3, II-4, II-5,and II-8), one uncle (II-6),
and one cousin (III-5) were diagnosed with type 2 diabetes mellitus
(T2DM) at the age of 40, 40, 45, 50, 50, 30, 40, 40, 50, 40, and 28
years, respectively. The proband and his mother corrected the diagnosis
of MIDD until 2022. Although errors were observed in the classification
of diabetes in this family, we could still observe that all patients had
early-onset diabetes and a few of them (I-2, II-1, and II-8) could
achieve glycemic control with oral hypoglycemic drugs alone. In addition
to the proband (III-1), his grandmother (I-2), grandaunt (I-3),
granduncle (I-4), mother (II-1), and two aunts (II-4 and II-8) had
hearing impairment, and his mother stated that the disease onset was
very early. Only the proband (III-1), his grandmother (I-2), and his
mother (II-1) had myoclonus, but some patients had diabetes alone (II-2,
II-3, II-5, II-6, and III-5). Combined symptoms suggested that the
occurrence of diabetes in this family was distinctive, and this type of
diabetes was inherited maternally, often together with other symptoms,
but the disease phenotype was often different.

\textbf{Table 1.} Physical and laboratory examination findings of the
proband\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
Year & 2013 & 2014 & 2019 & 2021 & 2022\tabularnewline
Symptoms & & & & &\tabularnewline
BMI, kg/m\textsuperscript{2} & 17.30 & 17.30 & 17.30 & 17.30 &
17.99\tabularnewline
Random urine glucose & Unknown & 3+ & 3+ & 1+ & 4+\tabularnewline
Ketones in urine & Unknown & 3+ & 2+ & 2+ & Neg\tabularnewline
Fasting plasma glucose, mmol/L & - & 6.95 & - & - & 10.60\tabularnewline
Fasting C-peptide, ng/mL & - & 0.6 & - & - & 0.32\tabularnewline
2-h fasting C-peptide, ng/mL & - & 3.0 & - & - & 0.63\tabularnewline
HbA1c, \% & 10.9 & 13.1 & 15.4 & 12.8 & 11.8\tabularnewline
Albumin, g/L & Normal & Normal & Normal & Normal & 38.6\tabularnewline
Globulin, g/L & Normal & Normal & Normal & Normal & 25.2\tabularnewline
Alanine aminotransferase, U/L & Normal & Normal & Normal & Normal &
11.3\tabularnewline
Aspartate aminotransferase, U/L & Normal & Normal & Normal & Normal &
11.1\tabularnewline
Creatinine, \selectlanguage{greek}μ\selectlanguage{english}mol/L & Normal & Normal & Normal & 137.0 &
69.0\tabularnewline
Uric acid, \selectlanguage{greek}μ\selectlanguage{english}mol/L & Normal & 441.4 & 595.3 & 648.0 & 424\tabularnewline
TG, mmol/L & Normal & Normal & 3.11 & 5.00 & 1.88\tabularnewline
TC, mmol/L & Normal & Normal & Normal & 6.38 & 3.77\tabularnewline
HDL-C, mmol/L & Normal & Normal & 0.83 & Normal & 1.00\tabularnewline
LDL-C, mmol/L & Normal & Normal & Normal & 3.125 & 2.22\tabularnewline
CRP, mg/L & - & Normal & - & Normal & 0.03\tabularnewline
Anti-GAD & - & - & - & - & Neg\tabularnewline
Anti-IAA & - & - & - & - & Neg\tabularnewline
Drug therapy & Lispro mix 25+Acarbose & BIAsp30 & BIAsp30 &
Apidra+IDegAsp +Voglibose & IDegAsp+Acarbose\tabularnewline
\bottomrule
\end{longtable}

The results of biochemical indicators when the proband was admitted to
the hospital. -: no detection; unknown: tests were conducted but the
results are unknown.

Abbreviations: BMI, body mass index; HbA1c, glycosylated hemoglobin;
HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density
lipoprotein cholesterol; TG, triglyceride; TC, total cholesterol; CRP,
C-reactive protein; GAD, glutamic acid decarboxylase antibody; IAA,
insulin autoantibodies; Neg, negative; +, positive.

\textbf{Table 2.} Disease characteristics of different patients in the
family\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
& Age at onset (years) & Gender & Symptom & Therapies &
Ending\tabularnewline
I-2 & 40 & F & Diabetes, lean, hearing impairment, myoclonus & OAD &
Alive\tabularnewline
I-3 & 40 & F & Diabetes, hearing impairment & Unknown &
Dead\tabularnewline
I-4 & 45 & M & Diabetes, hearing impairment & Unknown &
Dead\tabularnewline
II-1 & 50 & F & Diabetes, lean, hearing impairment, myoclonus & OAD &
Alive\tabularnewline
II-2 & 50 & F & Diabetes & Insulin injection & Alive\tabularnewline
II-3 & 30 & F & Diabetes & Insulin injection & Alive\tabularnewline
II-4 & 40 & F & Diabetes, hearing impairment & Insulin injection &
Alive\tabularnewline
II-5 & 40 & F & Diabetes & Unknown & Dead\tabularnewline
II-6 & 40 & M & Diabetes & Unknown & Dead\tabularnewline
II-8 & 50 & F & Diabetes, hearing impairment & OAD &
Alive\tabularnewline
III-1 & 23 & M & Diabetes, lean, hearing impairment, myoclonus & Insulin
injection & Alive\tabularnewline
III-5 & 28 & M & Diabetes & Insulin injection & Alive\tabularnewline
\bottomrule
\end{longtable}

Abbreviations: OAD, oral antidiabetic drug.

\textbf{Genetic analysis}

Depending on the clinical symptoms and maternal clustering
characteristic in the proband's family, we performed sequencing analysis
of the whole mtDNA in the proband. The proband exclusively had a
heteroplasmic m.3243A\textgreater{}G mutation in the MT-TL1, with a
mutation frequency of 31.46\% (Figure 2a and Table 3). This mutation was
detected in his mother (II-1) but absent in the other two family members
(his daughters IV-1 and IV-2) through Sanger sequencing (Figure 2b, 2c,
and 2d). Genetic testing confirmed that the aforementioned family
members belonged to the MIDD subtype.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2.} Sanger sequencing chromatogram. Arrows indicate the
changed position of the mutation in the MT-TL1 gene. (a) Proband
(III-1), mutant type; (b) the proband's mother (II-1), mutant type; (c)
the proband's daughter (IV-1), wild type; and (d) the proband's daughter
(IV-2), wild type;

\textbf{Table 3.} Detection details of the proband
mt.3243A\textgreater{}G mutation site\selectlanguage{english}
\begin{longtable}[]{@{}llllllll@{}}
\toprule
Chr & Mutation & Gene & Group & Homo/hete & sum\_depth & alt\_depth &
alt\_rate\tabularnewline
\midrule
\endhead
ChrM & m.3243A\textgreater{}G & MT-TL1 & tRNA & hete & 5000 & 1573 &
34.16\%\tabularnewline
\bottomrule
\end{longtable}

\textbf{Precise treatment based on the patient's genotype}

In this study, a patient had a re-diagnosis of MIDD after 10 years'
suffering, and an m.3243A\textgreater{}G mutation was identified in the
MT-TL1 gene in his family. Although the diagnosis is quite clear, the
clinical management of MIDD remains a complicated issue. This
information, combined withthe results reported in the literature, led to
the recommendation that everybody in his family needed individual-based
management. At this condition, the patient himself needed insulin
administration because he had defective insulin secretion by pancreatic
\selectlanguage{greek}β-\selectlanguage{english}cells with m.3243A\textgreater{}G mutant mtDNA. According to the
literature, we added coenzyme Q10 to improve themitochondrial function,
and drugs that may weaken mitochondrial function, such as metformin and
statins, had to be avoided. During follow-up, the incidence of diabetic
ketoacidosis was found to be significantly reduced, and fasting blood
glucose and HbA1c levels evidently improved. Cochlear implantation was
recommended for deafness. For maternal relatives of patients with MIDD,
long-term follow-up is advisable and a careful examination of mutant
mtDNA is preferred.

\textbf{Discussion}

MIDD is a group of clinical heterogeneous diseases caused by dysfunction
of the mitochondrial respiratory chain and oxidative phosphorylation,
and it manifests as multiorgan damage (\emph{8} ). MIDD often causes
early-onset diabetes and sensorineural deafness, but various other
organs systems are involved, including ophthalmic disease (\emph{9, 10}
), myopathy, encephalopathy, cardiac disease, gastrointestinal diseases
(\emph{11} ), renal disease (\emph{12} ), and so on. Because of
mitochondrial DNA heteroplasmy, a great difference in clinical
manifestations is observed among patients with MIDD. Even the same
mutation can exsit in different phenotypes or can be``silenced''
eternally. Although the mutation was screened only in four individuals
and two people were found to carry the mutation in this family, based on
the genetic characteristics of MIDD, we can reasonably suspect that
other people with diabetes carry the mutation, and asymptomatic carriers
cannot be ruled out. Of the 12 people with diabetes in our study, only
three were males. Seven patients had hearing impairment, three presented
myoclonus, three were lean, and five had diabetes only. In this family,
diabetes started at the age approximately 40 years, and for a few
people, the onset age was earlier. According to his mother, the disease
onset was very early. In this family, the proband (III-1), his
grandmother (I-2), and his mother (II-1) had myoclonus. The proband,
whom we did not encounter reported that, myoclonus always occurred in
the lower extremities, less frequently, and often accompanied by pain
after exercises, this symptom is consistent with that reported in the
literature. The patient had a correct diagnosis of the disease through
genetic technology after 10 years. The proband had a diagnosis of T1DM
because of his younger onset age (\textless{}30 years old), lean body,
poor glycemic control, low fasting C-peptide level, and repeated
ketoacidosis. The more important reason was that his hearing impairment
was progressive, so much so that when the patient was repeatedly
hospitalized for life-threatening ketoacidosis, his hearing impairment
was ignored by both the patient and the physician. Through careful
inquiry of the patient's history and physical examination, the onset
time of hearing problem and diabetes was found to be the same, and many
of the maternal relatives experienced the same situation. Further
genetic testing confirmed our suspicion that the patient had MIDD with
mt.3243A\textgreater{}G mutation. In conclusion, the clinical features
of MIDD are as follows: (1) maternal inheritance. Almost all offspring
of female patients carry the mutation, but not all of them are affected
by the disease. The offspring of male patients generally do not carry
this mutation. (2) A vast majority of people have diabetes at onset age
before 40 years, and most of them have diabetes onset in adulthood,
rarely in adolescents. (3) Most patients had normal or lean body mass,
with a mean BMI of less than 20 kg/m\textsuperscript{2}. (4) Mostly
negative results were obtained for islet autoantibodies. (5)
Abnormalities in glucose metabolism combined with multiorgan damage.
Special attention should be paid to the occurrence of early-onset
diabetic retinopathy in the proband. According to literature, the most
common ophthalmic feature is typical macular retinal dystrophy(\emph{13}
). Most patients with macular disease have a history of diabetes for
more than 5 years and the onset is always after the age of 40 years.
Early-onset diabetes and poor glycemic control may contribute to
diabetic retinopathy progression.

During a suspected diagnosis of MIDD in the clinical setting, the
diagnosis needs to be clearly confirmed by genetic testing such as
Sanger sequencing, gene chip sequencing, and high-throughput gene
sequencing. Blood leukocytes are mostly used for detection, but compared
with blood leukocytes, muscle, urine(\emph{13} ) and buccal
mucosa(\emph{14} ) are better as testing samples because of a higher
heterogeneity. Reports have suggested that the patient had an apparent
phenotype and family history, and the family carried the
mt.3243A\textgreater{}G mutation, but the blood test results of the
patient showed no mutation, which may be attributed to the heterogeneity
in the specimen.

In addition to the clinical phenotype, heteroplasmy plays an important
role in considering the severity of MIDD (\emph{15} ).
Mt.3243A\textgreater{}G occurs in a wide range of mitochondrial
encephalomyopathies including MELAS (mitochondrial myopathy,
encephalopathy, lactic acidosis, and stroke) or MELAS/MERRF (myoclonic
epilepsy and red ragged fibers), and overlap syndrome (\emph{16} ). The
proband's grandaunt (I-3), granduncle (I-4), one aunt (II-5), and one
uncle (II-6) died ofdiabetic complications. Whether the decreased
patients had these severe phenotypes leading to death is an open
question. Compared with his mother, the proband had an earlier onset age
and severe disease, and the same mutation (A3243G) was observed.
Considering a large-scale study in Finland, A3243G levels may gradually
increase over subsequent generations (\emph{17} ), we suggest that the
heterogeneous mutation frequency in his mother is lower than that in the
proband(34.16\%). The mt.A3243G heterogeneity level is negatively
correlated with age, which suggests that his mother has a lower
heterogeneous mutation frequency than the proband (\emph{18} ). A
meta-analysis showed that mitochondrial heterogeneity of A 3243G is
significantly associated with the incidence of diabetes, that is, the
greater the heterogeneity, the greater the probability of MIDD
development, and the younger the age of onset in offspring (\emph{19} ).
Our clinicians should note that genetic counseling plays a crucial part
in such a family, which can help asymptomatic patients achieve early
diagnosis and prevent complications. Unfortunately, we only screened the
mt.3243A\textgreater{}G mutation in the patient's mother and children.
His other family members should be urged to screen this mutation owing
to special inheritance. Early screening of mutation sites, especially
for patients with low mutation heterozygosity, may improve treatment and
prognosis and delay the occurrence of complications.

Because of the heterogeneity of MIDD, treatment for MIDD is highly
challenging. Patients with MIDD tend to be lean and have insufficient
energy synthesis; hence, they are unsuitable for a too strict diet to
avoid malnutrition or aggravation of the disease process. Endurance
training can lead to a decrease the proportion of the mutant
mitochondrial DNA; therefore, an appropriate level of exercise can
improve mitochondrial function (\emph{19} ). However, too strenuous
exercise can easily lead to severe lactic acidosis, a fatal condition,
in these patients. As for drug therapy, the most important aspect to
remember is that individualized treatment should be provided. Among the
patients in this family, five were treated with insulin and three were
treated with oral hypoglycemic drugs. The use of different glycemic
control regimens may be attributed to the different mutation load in
each patient. Although some patients do not need hypoglycemic agents
temporarily or need only oral hypoglycemic agents, we still recommend
starting insulin therapy as early as possible because mtDNA mutations
may aggravate \selectlanguage{greek}β-\selectlanguage{english}cell apoptosis to impair insulin secretion. As mentioned
above, we added coQ10 (\emph{20} ) to our patient during follow-up,
which proved that the drug could improve mitochondrial energy
metabolism. Other drugs can also enhance mitochondrial function, such as
lipoic acid (\emph{21} ) and thiamine. On the contrary, many common
drugs should be avoided in these patients, such as tetracycline,
chloramphenicol, sodium valproate, phenytoin, phenobarbital (\emph{22}
), antiretrovirals (\emph{23} ), statins, and metformin (\emph{24} ).
Because of the rapid progression of diabetic vascular complications in
the proband, we specifically advised the patient not to use statins to
avoid acute complications, and patients taking oral hypoglycemic drugs
were instructed to avoid metformin. However, we need to know that the
above treatment was administered only to control symptoms, and only gene
therapy is the fundamental treatment. Recombinant RNA molecules have
been designed such that they are imported into the mitochondria of human
cells to decrease the proportion of the mutant mitochondrial DNA
(\emph{25} ). However, this technique has not been applied to clinical
practice, necessitating further exploration.

In conclusion, MIDD is a complex syndrome with variable clinical
features and is often misdiagnosed. Our findings indicate that whole
mtDNA sequencing analysis can be used to detect m.3243A\textgreater{}G
mutation in the mtDNA. In clinical practice, the patient's medical
history, especially accompanying symptoms and family history, should
always be carefully recorded. Once the patient has a suspected
diagnosis, genetic testing should be performed as soon as possible to
make a timely diagnosis and standardize individualized treatment.

\textbf{Data Availability}

The data used to support the findings of this study are available from
the corresponding author upon request.

\textbf{Conflicts of Interest}

The authors declare that there are no conflicts of interest.

\textbf{Authors' Contributions}

Shasha Zheng and Juanjuan Wang contributed equally to this work. Wei Shi
and Zhongzhi Zhang gathered patient medical records from outside
hospitals. Hongmei Zhang and Zhongjing Wang conceived and designed the
experiments. Pei Wang, Minxian Sun, Juanjuan Wang and Shasha Zheng
analyzed the data. Shasha Zheng and Juanjuan Wang wrote the paper.

\textbf{Acknowledgments}

This study was supported by grants from the Key Research and development
Projects of Hubei Provincial Department of Science and Technology
(2020BHB018) , from the Wuhan Science and Technology Bureau Knowledge
Innovation Special Project (2023020201010187) and from the Beijing
Medical Award Foundation Project (YXJL-2023-Q227-2094).

\textbf{References}

1. Y. S. Ng \emph{et al.} , Mitochondrial disease in adults: recent
advances and future promise. \emph{Lancet Neurol} \textbf{20} , 573-584
(2021).

2. R. Murphy, D. M. Turnbull, M. Walker, A. T. Hattersley, Clinical
features, diagnosis and management of maternally inherited diabetes and
deafness (MIDD) associated with the 3243A\textgreater{}G mitochondrial
point mutation. \emph{Diabet Med} \textbf{25} , 383-399 (2008).

3. M. Yang \emph{et al.} , The Mutations and Clinical Variability in
Maternally Inherited Diabetes and Deafness: An Analysis of 161
Patients.\emph{Front Endocrinol (Lausanne)} \textbf{12} , 728043 (2021).

4. J. M. van den Ouweland \emph{et al.} , Mutation in mitochondrial
tRNA(Leu)(UUR) gene in a large pedigree with maternally transmitted type
II diabetes mellitus and deafness. \emph{Nat Genet} \textbf{1} , 368-371
(1992).

5. M. Tabebi \emph{et al.} , The first concurrent detection of
mitochondrial DNA m.3243A\textgreater{}G mutation, deletion, and
depletion in a family with mitochondrial diabetes. \emph{Mol Genet
Genomic Med} \textbf{8} , e1292 (2020).

6. W. Jia \emph{et al.} , Standards of medical care for type 2 diabetes
in China 2019. \emph{Diabetes Metab Res Rev} \textbf{35} , e3158 (2019).

7. A. American Diabetes, 2. Classification and Diagnosis of Diabetes:
Standards of Medical Care in Diabetes-2021. \emph{Diabetes
Care}\textbf{44} , S15-S33 (2021).

8. H. Sun \emph{et al.} , IDF Diabetes Atlas: Global, regional and
country-level diabetes prevalence estimates for 2021 and projections for
2045. \emph{Diabetes Res Clin Pract} \textbf{183} , 109119 (2022).

9. E. Kisilevsky, P. Freund, E. Margolin, Mitochondrial disorders and
the eye. \emph{Surv Ophthalmol} \textbf{65} , 294-311 (2020).

10. N. Oishi \emph{et al.} , Multimodal imaging analysis of macular
dystrophy in patient with maternally inherited diabetes and deafness
(MIDD) with m.3243A\textgreater{}G mutation. \emph{Ophthalmic
Genet}\textbf{42} , 304-311 (2021).

11. M. Karppa, I. Mahjneh, A. Karttunen, U. Tolonen, K. Majamaa, Muscle
computed tomography patterns in patients with the mitochondrial DNA
mutation 3243A\textgreater{}G. \emph{J Neurol} \textbf{251} , 556-563
(2004).

12. P. J. Guillausseau \emph{et al.} , Heterogeneity of diabetes
phenotype in patients with 3243 bp mutation of mitochondrial DNA
(Maternally Inherited Diabetes and Deafness or MIDD). \emph{Diabetes
Metab} \textbf{30} , 181-186 (2004).

13. R. G. Coussa, S. Parikh, E. I. Traboulsi, Mitochondrial DNA A3243G
variant-associated retinopathy: Current perspectives and clinical
implications. \emph{Surv Ophthalmol} \textbf{66} , 838-855 (2021).

14. S. Shanske \emph{et al.} , Varying loads of the mitochondrial DNA
A3243G mutation in different tissues: implications for
diagnosis.\emph{Am J Med Genet A} \textbf{130A} , 134-137 (2004).

15. K. N. Robinson, S. Terrazas, S. Giordano-Mooga, N. A. Xavier, The
Role of Heteroplasmy in the Diagnosis and Management of Maternally
Inherited Diabetes and Deafness. \emph{Endocr Pract} \textbf{26} ,
241-246 (2020).

16. M. Michaelides \emph{et al.} , Macular dystrophy associated with the
A3243G mitochondrial DNA mutation. Distinct retinal and associated
features, disease variability, and characterization of asymptomatic
family members. \emph{Arch Ophthalmol} \textbf{126} , 320-328 (2008).

17. W. Wei, P. F. Chinnery, Inheritance of mitochondrial DNA in humans:
implications for rare and common diseases. \emph{J Intern
Med}\textbf{287} , 634-644 (2020).

18. S. Zhang \emph{et al.} , Heteroplasmy level of the mitochondrial
tRNaLeu(UUR) A3243G mutation in a Chinese family is positively
associated with earlier age-of-onset and increasing severity of
diabetes. \emph{Chin Med Sci J} \textbf{24} , 20-25 (2009).

19. A. W. El-Hattab, A. M. Zarante, M. Almannai, F. Scaglia, Therapies
for mitochondrial diseases and current clinical trials. \emph{Mol Genet
Metab} \textbf{122} , 1-9 (2017).

20. R. H. Haas, The evidence basis for coenzyme Q therapy in oxidative
phosphorylation disease. \emph{Mitochondrion} \textbf{7 Suppl} ,
S136-145 (2007).

21. M. Wang \emph{et al.} , A Deafness- and Diabetes-associated tRNA
Mutation Causes Deficient Pseudouridinylation at Position 55 in tRNAGlu
and Mitochondrial Dysfunction. \emph{J Biol Chem} \textbf{291} ,
21029-21041 (2016).

22. C. W. Lam, C. H. Lau, J. C. Williams, Y. W. Chan, L. J. Wong,
Mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like
episodes (MELAS) triggered by valproate therapy. \emph{Eur J
Pediatr}\textbf{156} , 562-564 (1997).

23. G. Moyle, Mitochondrial toxicity: myths and facts. \emph{J HIV
Ther}\textbf{9} , 45-47 (2004).

24. H. Ninomiya \emph{et al.} , Treatment of Mitochondrial Diabetes with
a Peroxisome Proliferator-activated Receptor (PPAR)-gamma
Agonist.\emph{Intern Med} \textbf{55} , 1143-1147 (2016).

25. Y. Tonin \emph{et al.} , Modeling of antigenomic therapy of
mitochondrial diseases by mitochondrially addressed RNA targeting a
pathogenic point mutation in mitochondrial DNA. \emph{J Biol
Chem}\textbf{289} , 13323-13334 (2014).

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