3670 PART 16 Genes, the Environment, and Disease

causing Kearns-Sayre syndrome (KSS) and the discovery of a point

mutation in ND4, an mtDNA-encoded complex I gene, causing Leber’s

hereditary optic neuropathy (LHON). Following these two discoveries,

>400 pathogenic mtDNA mutations or deletions have been reported to

cause human disease.

MITOCHONDRIAL DNA STRUCTURE AND

FUNCTION

As a result of its circular structure and extranuclear location, the replication and transcription mechanisms of mtDNA differ from the corresponding mechanisms in the nuclear genome, whose nucleosomal

packaging and structure are more complex. Specifically, mitochondria

have their own transcription system, and the mtDNA itself replicates

independently of cellular replication. Because each cell contains many

copies of mtDNA and because the number of mitochondria can vary

during the lifetime of each cell, mtDNA copy number is not directly

coordinated with the cell cycle. Thus, vast differences in mtDNA

copy number are observed between different cell types and tissues

and during the lifetime of a cell. Another important feature of the

mtDNA replication process is a reduced stringency of proofreading

and replication error correction, leading to a greater degree of sequence

variation compared to the nuclear genome. Some of these sequence

variants are silent polymorphisms that do not have the potential for

a phenotypic or pathogenic effect, whereas others may be considered

pathogenic mutations. There are some mutations that may be considered ecogenetic, as they typically remain silent, meaning they do not

cause disease, unless an external event occurs. One classic example is

seen in a common (1:800) mutation in the mitochondrial 12S rRNA

gene, m.A1555G, which is associated with hearing loss but is rapidly

exacerbated by exposure to normal dosages of an aminoglycoside.

Because mtDNA replication is independent of cellular replication, the

percentage of mutant mtDNA copies tend to increase with age in cells

that are terminally differentiated (nonreplicative) at birth such as neurons and myocytes, which may explain some features of mitochondrial

dysfunction with aging.

With respect to transcription, initiation can occur on both strands

and proceeds through the production of an intronless polycistronic

precursor RNA, which is then processed to produce the 13 individual

mRNA and 24 individual tRNA and rRNA products. The 37 mtDNA

genes comprise fully 93% of the 16,569 nucleotides of the mtDNA

in what is known as the coding region. The control region, which is

contained in the D-loop, consists of ~1.1 kilobases (kb) of noncoding

DNA and is thought to have an important role in replication and transcription initiation.

■ MATERNAL INHERITANCE AND LACK OF

RECOMBINATION

In contrast to homologous pair recombination that takes place in the

nucleus, mtDNA molecules do not undergo recombination, such that

mutational events represent the only source of mtDNA genetic diversification. Moreover, it is only the maternal DNA that is transmitted to

the offspring. The fertilized oocyte degrades mtDNA carried from the

sperm in a complex process involving the ubiquitin proteasome system

and autophagy that takes place on the inner membrane of the oocyte.

Thus, although mothers transmit their mtDNA to both their sons and

daughters, only the daughters can transmit the inherited mtDNA to

future generations. Accordingly, mtDNA sequence variation and associated phenotypic traits and diseases are inherited exclusively along

maternal lines, meaning both sons and daughters have equal chances of

having symptomatic disease, with the only significant exception being

LHON, as described below.

The phenotypic expression, including age of onset and the exact

pattern of organ dysfunction, of a pathogenic mtDNA mutation may

vary greatly, even within families. Because of this complex relationship

between mtDNA mutations and disease expression, sometimes it is difficult to recognize the maternal pattern of inheritance at the clinical or

pedigree level. However, evidence of paternal transmission can almost

certainly exclude an mtDNA genetic origin of phenotypic variation or

disease; conversely, a disease affecting both sexes without evidence of

paternal transmission strongly suggests a heritable mtDNA disorder

(Fig. 468-2).

■ MULTIPLE COPY NUMBER (POLYPLOIDY), HIGH

MUTATION RATE, HETEROPLASMY, AND MITOTIC

SEGREGATION

Each aerobic cell in the body has multiple mitochondria, often numbering many hundreds or more in cells with extensive energy production requirements. Furthermore, the number of copies of mtDNA

within each mitochondrion varies from several to hundreds; this is

true of both somatic as well as germ cells, including oocytes in females.

In the case of somatic cells, this means that the impact of most newly

acquired somatic mtDNA mutations is likely to be very small in terms

of total cellular or organ system function; however, because of the

manyfold higher mutation rate during mtDNA replication, numerous

different mutations may accumulate with aging of the organism. It has

been proposed that the total cumulative burden of acquired somatic

mtDNA mutations with age may result in an overall perturbation of

mitochondrial function, contributing to age-related reduction in the

efficiency of oxidative phosphorylation and increased production of

damaging ROS. Because mtDNA (and nDNA) mutations may result

in electron leak within the ETC, the ROS damage may rise above the

normal baseline in some with specific mutations, resulting in increased

susceptibility to somatic mtDNA damage and disease expression. The

accumulation of such acquired somatic mtDNA mutations with aging

may contribute to age-related diseases, such as metabolic syndrome

and diabetes, cancer, and neurodegenerative and cardiovascular disease in any given individual. However, somatic mutations are not

carried forward to the next generation, and the hereditary impact of

mtDNA mutagenesis requires separate consideration of events in the

female germline.

The multiple mtDNA copy number within each cell, including the

maternal germ cells, results in the phenomenon of heteroplasmy, in

contrast to much greater uniformity (homoplasy) of somatic nuclear

DNA sequence. Heteroplasmy for a given mtDNA sequence variant

or mutation arises as a result of the coexistence within a cell, tissue, or

individual of mtDNA molecules bearing more than one version of the

sequence variant (Fig. 468-3). The importance of the heteroplasmy

phenomena to the understanding of mtDNA-related mitochondrial

diseases is critical. The coexistence of mutant and nonmutant (wildtype) mtDNA and the variation of the mutant load, which can be

thought of as the percentage of mutant mtDNA molecules within a

specific cell, tissue, organ, or organism, contribute to the expression of

a phenotype among individuals from the same maternal sibship. At the

level of the oocyte, the percentage of mtDNA molecules bearing each

version of the polymorphic sequence variant or mutation depends on

stochastic events related to partitioning of mtDNA molecules during

the process of oogenesis itself. Thus, oocytes differ from each other

in the degree of heteroplasmy for that sequence variant or mutation.

In turn, the heteroplasmic state is carried forward to the zygote and

to the organism as a whole, to varying degrees, depending on mitotic

segregation of mtDNA molecules during organ system development

and maintenance. For this reason, in vitro fertilization, followed by

preimplantation genetic diagnosis (PGD), is not as predictive of the

genetic health of the offspring in the case of mtDNA mutations as in

the case of mutations and subsequent diseases occurring in the nuclear

genome. Similarly, the impact of somatic mtDNA mutations acquired

during development and subsequently also shows an enormous spectrum of variability. In general, a higher mutant load will result in a

more severe and earlier phenotypic presentation. However, measuring

heteroplasmy in one tissue (lymphocytes from blood or urine sediment

containing kidney and bladder epithelial cells, for example) may not

represent the percentage of mutant heteroplasmy in the tissue or organs

most affected, such as the cardiac atrioventricular node or brain. Furthermore, the threshold of mutant heteroplasmy that results in clinical

illness may vary depending on the specific mutation.

Mitotic segregation refers to the unequal distribution of wild-type

and mutant versions of mtDNA molecules during all cell divisions

that occur during prenatal development and subsequently throughout

Mitochondrial DNA and Heritable Traits and Diseases

3671CHAPTER 468

Oocyte maturation

and mtDNA amplification Fertilization

Mature oocytes

Primary oocytes

Mutant mitochondrion

Normal mitochondrion

Nucleus

Primordial germ

cell containing

mutant mtDNA

High level of mutation

(affected offspring)

Intermediate level

of mutation (mildly

affected offspring)

Low level of mutation

(unaffected offspring)

FIGURE 468-3 Heteroplasmy and the mitochondrial genetic bottleneck. During the production of

primary oocytes, a selected number of mitochondrial DNA (mtDNA) molecules are transferred into

each oocyte. Oocyte maturation is associated with the rapid replication of this mtDNA population.

This restriction-amplification event can lead to a random shift of mtDNA mutational load between

generations and is responsible for the variable levels of mutated mtDNA observed in affected

offspring from mothers with pathogenic mtDNA mutations. Mitochondria that contain mutated

mtDNA are shown in red, and those with normal mtDNA are shown in green. (Reproduced with

permission from R Taylor, D Turnbull: Mitochondrial DNA mutations in human disease. Nat Rev

Genetics 6:389, 2005.)

the lifetime of an individual. The phenotypic effect or disease impact

will be a function not only of the inherent disruptive effect (pathogenicity) on the mtDNA-encoded gene (coding region mutations) or

integrity of the mtDNA molecule (control region mutations) but also

of its distribution among the multiple copies of mtDNA in the various

mitochondria, cells, and tissues of the affected individual. Thus, one

consequence can be the generation of a bottleneck due to the marked

decline in given sets of mtDNA variants, pathogenic and nonpathogenic, consequent to such mitotic segregation. It is postulated that the

main effects of this bottleneck occur between the primordial germ

cell state and the primary oocyte stage of development. Heterogeneity

arises from differences in the degree of heteroplasmy among oocytes of

the transmitting female, together with subsequent, probably random,

mitotic segregation of the pathogenic mutation during tissue and organ

development and throughout the lifetime of the individual offspring.

The actual expression of disease might then depend on a threshold

percentage of mitochondria whose function is disrupted by mtDNA

mutations. This in turn confounds hereditary transmission patterns

and hence genetic diagnosis of pathogenic heteroplasmic mutations.

Generally, if the proportion of mutant mtDNA is <60%, the individual

is unlikely to be affected, whereas proportions exceeding 90% cause

clinical disease. One notable exception is LHON, in which these mutations are present either in 100% mutant homoplasmy, which causes the

disease expression, or 100% wild-type homoplasmy. It is not understood why this specific phenotype and the several known mtDNA

alleles that result in LHON behave in this manner.

■ HOMOPLASMIC VARIANTS AND HUMAN mtDNA

PHYLOGENY

In contrast to classic mtDNA diseases, most of which begin in childhood

and are the result of heteroplasmic mutations as noted above, during

the course of human evolution, certain mtDNA sequence variants have

drifted to a state of homoplasmy, wherein all of the mtDNA molecules

in the organism contain the new sequence variant. This arises due to a

“bottleneck” effect followed by genetic drift during the very process of

oogenesis itself (Fig. 468-3). In other words, during certain stages of

oogenesis, the mtDNA copy number becomes so substantially reduced

that the particular mtDNA species bearing the novel or derived

sequence variant may become the increasingly predominant, and eventually exclusive, version of the mtDNA for

that particular nucleotide site. All of the offspring of a

woman bearing an mtDNA sequence variant or mutation

that has become homoplasmic will also be homoplasmic

for that variant and will transmit the sequence variant

forward in subsequent generations.

Considerations of reproductive fitness limit the evolutionary or population emergence of pathogenic homoplasmic mutations that are lethal or cause severe disease

in infancy or childhood. Thus, with a number of notable

exceptions (e.g., mtDNA mutations causing LHON; see

below), most homoplasmic mutations are considered to

be neutral markers of human evolution, which are useful

and interesting in the population genetics analysis of

shared maternal ancestry but have little significance in

human phenotypic variation or disease predisposition.

More important is the understanding that this accumulation of homoplasmic mutations occurs at a genetic

locus that is transmitted only through the female germline and that lacks recombination. In turn, this enables

reconstruction of the sequential topology and radiating

phylogeny of mutations accumulated through the course

of human evolution since the time of the most recent

common mtDNA ancestor of all contemporary mtDNA

sequences, some 200,000 years ago. The term haplogroup

is usually used to define major branching points in

the human mtDNA phylogeny, nested one within the

other, which often demonstrate striking continental geographic ancestral partitioning. At the level of the complete mtDNA sequence, the term haplotype is usually

used to describe the sum of mutations observed for a given mtDNA

sequence and as compared to a reference sequence, such that all haplotypes falling within a given haplogroup share the total sum of mutations that have accumulated since the most recent common ancestor

and the bifurcation point they mark. The remaining observed variants

are private to each haplotype. Consequentially, the human mtDNA

sequence is an almost perfect molecular prototype for a nonrecombining locus, and its variation has been extensively used in phylogenetic

studies. Moreover, the mtDNA mutation rate is considerably higher

than the rate observed for the nuclear genome, especially in the control region, which contains the displacement loop, or D-loop, in turn

comprising two adjacent hypervariable regions (HVR-I and HVR-II).

Together with the absence of recombination, this amplifies drift to

high frequencies of novel haplotypes. As a result, mtDNA haplotypes

are more highly partitioned across geographically defined populations

than sequence variants in other parts of the genome. Despite extensive

research, it has not been well established that such haplotype-based

partitioning has a significant influence on human health conditions.

However, mtDNA-based phylogenetic analysis can be used both as a

quality assurance tool and as a filter in distinguishing neutral mtDNA

variants comprising human mtDNA phylogeny from potentially deleterious mutations.

MITOCHONDRIAL DNA DISEASE

The true prevalence of mtDNA disease is difficult to estimate because

of the phenotypic heterogeneity that occurs as a function of heteroplasmy, the challenge of detecting and assessing heteroplasmy in

different affected tissues, and the other unique features of mtDNA

function and inheritance described above. It is estimated that at least

1 in 200 healthy humans harbors a pathogenic mtDNA mutation with

the potential to causes disease but that heteroplasmic germline pathogenic mtDNA mutations actually result in clinical disease in ~1 in 5000

individuals.

The true disease burden relating to mtDNA sequence variation will

only be known when the following capabilities become available: (1)

ability to distinguish a completely neutral sequence variant from a true

phenotype-modifying or pathogenic mutation, (2) accurate assessment of heteroplasmy that can be determined with fidelity, and (3) a

3672 PART 16 Genes, the Environment, and Disease

Parkinsonism,

aminoglycoside-induced deafness

MELAS

myoglobinuria Myopathy,

PEO

Cardiomyopathy

ECM

Diabetes and deafness

Cardiomyopathy,

SIDS, ECM Cardiomyopathy,

PEO, MERRF,

MELAS, deafness

PPK, deafness,

MERRF-MELAS

PEO

PEO

Myopathy, lymphoma

Myopathy, MELAS

Myopathy,

cardiomyopathy, PEO

PEO, LHON, MELAS,

myopathy, cardiomyopathy,

diabetes and deafness

Cardiomyopathy

LS, MELAS,

multisystem disease

ECM

Myopathy,

PEO

LS, ataxia,

chorea, myopathy

Cardiomyopathy

myoclonus

Cardiomyopathy

ECM

Cardiomyopathy, ECM

PEO, myopathy,

sideroblastic anemia

ECM, LHON, myopathy,

cardiomyopathy, MELAS

and parkinsonism

LHON, MELAS,

diabetes,

LHON and dystonia

LS, MELAS

LHON, myopathy,

LHON and dystonia

LHON

LS, ECM,

myoglobinuria

NARP, MILS,

FBSN

Cardiomyopathy

LHON

LHON Cyt b

ND6

ND2 ND5

ND4

ND4L

ND3

COXIII

COXII

COXI

ND1

L1

16S

V 12sF

PT

E

H

R

K G

D

S1

Y

C

N

A

W

M

Q

I

L2

S2

A6

A8

Myoglobinuria,

motor neuron disease,

sideroblastic anemia

Myopathy,

multisystem disease,

encephalomyopathy

Progressive myoclonus,

epilepsy, and optic atrophy

FIGURE 468-4 Mutations in the human mitochondrial genome known to cause disease. Disorders that are frequently

or prominently associated with mutations in a particular gene are shown in boldface. Diseases due to mutations that

impair mitochondrial protein synthesis are shown in blue. Diseases due to mutations in protein-coding genes are shown

in red. ECM, encephalomyopathy; FBSN, familial bilateral striatal necrosis; LHON, Leber’s hereditary optic neuropathy;

LS, Leigh’s syndrome; MELAS, mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes; MERRF,

myoclonic epilepsy with ragged red fibers; MILS, maternally inherited Leigh’s syndrome; NARP, neuropathy, ataxia,

and retinitis pigmentosa; PEO, progressive external ophthalmoplegia; PPK, palmoplantar keratoderma; SIDS, sudden

infant death syndrome. (From S DiMauro, E Schon: Mitochondrial respiratory-chain diseases. N Engl J Med 348:2656,

2003. Copyright © 2003, Massachusetts Medical Society. Reprinted with permission from Massachusetts Medical

Society.)

systems biology approach (Chap. 486) to

determine the network of epistatic interactions of mtDNA sequence variations

with mutations in the nuclear genome.

■ OVERVIEW OF CLINICAL

AND PATHOLOGIC FEATURES

OF HUMAN mtDNA DISEASE

Given the vital roles of mitochondria

in all nucleated cells, it is not surprising that mtDNA mutations can affect

numerous tissues with pleiotropic

effects. More than 200 different diseasecausing, mostly heteroplasmic mtDNA

mutations have been described affecting

ETC function. Figure 468-4 provides

a partial mtDNA map of some of the

better characterized of these disorders.

A number of clinical clues can increase

the index of suspicion for a heteroplasmic mtDNA mutation as an etiology

of a heritable trait or disease, including

(1) familial clustering with absence of

paternal transmission; (2) adherence to

one of the classic syndromes (see below)

or paradigmatic combinations of disease

phenotypes involving several organ systems that normally do not fit together

within a single nuclear genomic mutation category; (3) a complex of laboratory and pathologic abnormalities that

reflect disruption in cellular energetics

(e.g., lactic acidosis and neurodegenerative and myodegenerative symptoms

with the finding of ragged red fibers,

reflecting the accumulation of abnormal mitochondria under the muscle sarcolemmal membrane); or (4) a mosaic

pattern reflecting a heteroplasmic state.

There are no truly sensitive and specific biomarkers of disease, and the

presence of a historically quintessential finding of ragged red fibers can

be seen in numerous muscle disorders, so laboratory tests must always

be interpreted in the context of their limitations and should not be used

to define the disease.

Heteroplasmy can sometimes be elegantly demonstrated at the

tissue level using histochemical staining for enzymes in the oxidative

phosphorylation pathway, with a mosaic pattern indicating heterogeneity of the genotype for the coding region for the mtDNA-encoded

enzyme. Complex II, CoQ, and cytochrome c are exclusively encoded

by nuclear DNA. In contrast, complexes I, III, IV, and V contain at least

some subunits encoded by mtDNA. Just 3 of the 13 subunits of the

ETC complex IV enzyme, cytochrome c oxidase (COX), are encoded

by mtDNA, and therefore, this enzyme has the lowest threshold for

dysfunction when a threshold level of mutated mtDNA is reached. Histochemical staining for COX activity in tissues of patients affected with

heteroplasmic inherited mtDNA mutations (or with the somatic accumulation of mtDNA mutations, see below) can show a mosaic pattern

of reduced histochemical staining in comparison with histochemical

staining for the complex II enzyme succinate dehydrogenase (SDH)

(Fig. 468-5). Heteroplasmy can also be detected at the genetic level

through direct Sanger-type mtDNA genotyping under special conditions, although clinically significant low levels of heteroplasmy can

escape detection in genomic samples extracted from whole blood using

conventional genotyping and sequencing techniques. Next-generation

sequencing (NGS) has largely overcome these limitations and enables

reliable detection and quantitation of heteroplasmy.

NGS has dramatically improved the clinical genetic diagnostic evaluation of mitochondrial diseases at the level of both the nuclear genome

and mtDNA. In the context of the larger nuclear genome, the ability

of NGS techniques to dramatically increase the speed at which DNA

can be sequenced at a fraction of the cost of conventional Sanger-type

sequencing technology is particularly beneficial. Low sequencing costs

and short turnaround time expedite “first-tier” screening of panels of

hundreds of previously known or suspected mitochondrial disease genes

or screening for the entire exome or genome in an attempt to identify

novel genes and mutations affecting different patients or families. In the

context of the mtDNA, NGS approaches now provide rapid and reliable

detection of heteroplasmy in different affected tissues. Although Sanger

sequencing allows for complete coverage of the mtDNA, it is limited by

the lack of deep coverage and low sensitivity for heteroplasmy detection

when levels are <50%. In contrast, NGS technology is an excellent tool

for rapidly and accurately obtaining a patient’s predominant mtDNA

sequence as well as lower frequency heteroplasmic variants and can

reliably detect mutant heteroplasmy <10%. Lower levels are often only

clinically relevant if in the setting of a striking difference in heteroplasmy

in different tissues. This capability to detect heteroplasmy at levels that

assist in pointing to an mtDNA-based disease process emanates from

deep coverage of the genome through multiple independent sequence

reads. Accordingly, recent studies making use of NGS techniques have

demonstrated sequence accuracy equivalent to Sanger-type sequencing

but also have uncovered heretofore unappreciated heteroplasmy rates

ranging between 10% and 50% and detection of single nucleotide heteroplasmy down to levels of <10%.

Clinically, the most striking overall characteristic of mitochondrial

genetic disease is the phenotypic heterogeneity associated with mtDNA

mutations. This extends to intrafamilial phenotypic heterogeneity

for the same mtDNA pathogenic mutation and, conversely, to the

overlap of phenotypic disease manifestations with distinct mutations.

Thus, although fairly consistent and well-defined “classic” syndromes

Mitochondrial DNA and Heritable Traits and Diseases

3673CHAPTER 468

FIGURE 468-5 Cytochrome c oxidase (COX) deficiency in mitochondrial DNA (mtDNA)–associated disease. Transverse

tissue sections that have been stained for COX and succinate dehydrogenase (SDH) activities sequentially, with

COX-positive cells shown in brown and COX-deficient cells shown in blue. A. Skeletal muscle from a patient with a

heteroplasmic mitochondrial tRNA point mutation. The section shows a typical “mosaic” pattern of COX activity, with

many muscle fibers harboring levels of mutated mtDNA that are above the crucial threshold to produce a functional

enzyme complex. B. Cardiac tissue (left ventricle) from a patient with a homoplasmic tRNA mutation that causes

hypertrophic cardiomyopathy, which demonstrates an absence of COX in most cells. C. A section of cerebellum from a

patient with mtDNA rearrangement that highlights the presence of COX-deficient neurons. D, E. Tissues that show COX

deficiency due to clonal expansion of somatic mtDNA mutations within single cells—a phenomenon that is seen in both

postmitotic cells (D; extraocular muscles) and rapidly dividing cells (E; colonic crypt) in aging humans. (Reproduced

with permission from R Taylor, D Turnbull: Mitochondrial DNA mutations in human disease. Nat Rev Genetics 6:389,

2005.)

have been attributed to specific mutations, frequently “nonclassical”

combinations of disease phenotypes ranging from isolated myopathy

to extensive multisystem disease are often encountered, rendering

genotype-phenotype correlation challenging. In both classical and

nonclassical mtDNA disorders, there is often a clustering of some combination of abnormalities affecting the neurologic system (including

optic nerve atrophy, pigment retinopathy, and sensorineural hearing

loss), cardiac and skeletal muscle (including extraocular muscles), and

endocrine and metabolic systems (including diabetes mellitus). Additional organ systems that may be affected include the hematopoietic,

renal, hepatic, and gastrointestinal systems, although these are more

frequently involved in infants and children. Disease-causing mtDNA

coding region mutations can affect either one of the 13 proteinencoding genes or one of the 24 protein synthetic genes. Clinical manifestations do not readily distinguish these two categories, although

lactic acidosis and specific muscle pathologic findings (e.g., ragged

red and ragged blue fibers, described immunohistochemical staining,

paracrystalline inclusions on ultrastructure) tend to be more prominent in the latter. In all cases, either defective ATP production due

to disturbances in the ETC or enhanced generation of ROS has been

invoked as the mediating biochemical mechanism between mtDNA

mutation and disease manifestation.

■ mtDNA DISEASE PRESENTATIONS

The clinical presentation of adult patients with mtDNA disease can

be divided into three categories: (1) clinical features suggestive of

mitochondrial disease (Table 468-2) but not a well-defined classic

syndrome; (2) classic mtDNA syndromes; and (3) clinical presentation

confined to one organ system (e.g., isolated sensorineural deafness,

cardiomyopathy, or diabetes mellitus).

Table 468-3 provides a summary of eight illustrative classic mtDNA

syndromes or disorders that affect adult patients and highlights some

of the most interesting features of mtDNA disease in terms of molecular pathogenesis, inheritance, and clinical presentation. The first five

of these syndromes result from heritable point mutations in either

protein-encoding or protein synthetic mtDNA genes; the other three

result from rearrangements or deletions

that usually do not involve the germline.

LHON is a common cause of maternally inherited visual failure. LHON typically presents during young adulthood

with subacute painless loss of vision in

one eye, with symptoms developing in

the other eye 6–12 weeks later. In some

instances, cerebellar ataxia, peripheral

neuropathy, and cardiac conduction

defects are observed. In >95% of cases,

LHON is due to one of the three homoplasmic point mutations of mtDNA that

affect genes encoding different subunits

of complex I of the mitochondrial ETC;

however, not all individuals who inherit

a primary LHON mtDNA mutation

develop optic neuropathy, and the maleto-female ratio is 8.2, indicating that

additional environmental (e.g., tobacco

exposure) or independent genetic factors are important in the etiology of the

disorder. Both the nuclear and mitochondrial genomic backgrounds modify

disease penetrance. Indeed, a region of

the X chromosome containing a highrisk haplotype for LHON has been

identified, supporting the formulation

that nuclear genes act as modifiers and

affording an explanation for the male

prevalence of LHON. This haplotype

can be used in predictive genomic testing

and prenatal screening for this disease.

In contrast to the other classic mtDNA disorders, it is of interest that

patients with this syndrome are often homoplasmic for the diseasecausing mutation. The somewhat later onset in young adulthood and

modifying effect of protective background nuclear genomic haplotypes

may have enabled homoplasmic pathogenic mutations to have escaped

evolutionary censoring.

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like

episodes (MELAS) is a multisystem disorder with a typical onset

between 2 and 10 years of age. Following normal early psychomotor

development, the most common initial symptoms are seizures, recurrent headaches, anorexia, and recurrent vomiting. Exercise intolerance

or proximal limb weakness can be the initial manifestation, followed

by generalized tonic-clonic seizures. Short stature is common. Seizures

are often associated with stroke-like episodes of transient hemiparesis

or cortical blindness that may produce recurrent encephalopathy with

impaired consciousness. It is often not possible to determine if the

encephalopathy is due to refractory clinical or subclinical seizures or

should be attributed to an independent effect. The cumulative residual

effects of the stroke-like episodes gradually impair motor abilities,

vision, and cognition, often by adolescence or young adulthood.

Sensorineural hearing loss adds to the progressive decline of these

TABLE 468-2 Common Features of Mitochondrial DNA–Associated

Diseases in Adults

Neurologic: stroke, epilepsy, migraine headache, peripheral neuropathy, ataxia,

dystonia, myoclonus, cranial neuropathy (optic atrophy, sensorineural deafness,

dysphagia, dysphasia)

Skeletal myopathy: ophthalmoplegia, exercise intolerance, myalgia, weakness

Cardiac: conduction block, cardiomyopathy

Respiratory: hypoventilation, aspiration pneumonitis

Endocrine: diabetes mellitus, premature ovarian failure, hypothyroidism,

hypoparathyroidism

Ophthalmologic: cataracts, pigment retinopathy, neurologic and myopathic (optic

atrophy, ophthalmoplegia)

3674 PART 16 Genes, the Environment, and Disease

individuals. A plethora of less common symptoms have been described

including myoclonus, ataxia, episodic coma, optic atrophy, cardiomyopathy, pigmentary retinopathy, ophthalmoplegia, diabetes mellitus,

hirsutism, gastrointestinal dysmotility, and nephropathy. The typical

age of death ranges from 10 to 35 years, but some individuals live into

their sixth decade. Intercurrent infections or intestinal obstructions are

often the terminal events. It is not atypical for some family members

to have much less severe or later onset illness, presumably because of

a lessor mutation load, and “MELAS” is not used as a diagnosis for

these restricted phenotypes. This creates somewhat of a disconnect

between the genotype for MELAS (most commonly the m.3243A>G

mutation) and a diverse phenotype, which includes the syndrome

MELAS, a syndrome of high-frequency hearing loss and diabetes with

onset later in life, as well as many other phenotypes between these two

extreme syndromes. Certain other mtDNA mutations can also cause

such patterns of diverse phenotypic expression. Laboratory investigation commonly demonstrates elevated lactate concentrations at rest

with excessive increase after moderate exercise. Brain imaging during

stroke-like episodes shows areas of involvement on T2- or fluid-attenuated inversion recovery (FLAIR) sequences, with decreased signal on

perfusion-weighted sequences, which typically involve the posterior

cerebrum and do not conform to the distribution of major arteries.

These magnetic resonance imaging (MRI) abnormalities may be temporary or evolve to subsequent atrophy (Fig. 468-6). Electrocardiography (ECG) may show evidence of cardiomyopathy, preexcitation, or

incomplete heart block. Electromyography and nerve conduction studies are consistent with a myopathic process, without or with coexisting

axonal and sensory neuropathic findings. Muscle biopsy typically

shows ragged red fibers with the modified Gomori trichrome stain or

“ragged blue fibers” with the SDH histochemical stain, resulting from

the hyperintense reaction. The diagnosis of MELAS is based on a combination of clinical findings and molecular genetic testing. Mutations

in the mtDNA gene MT-TL1 encoding tRNAleu are causative. The most

common mutation, present in ~80% of individuals with typical clinical

findings, is an A-to-G transition at nucleotide 3243 (m.3243A>G).

Mutations can usually be detected in mtDNA from leukocytes in individuals with typical MELAS; however, the occurrence of heteroplasmy

can result in varying tissue distribution of mutated mtDNA. In the

absence of specific treatment, various manifestations of MELAS are

treated according to standard modalities for prevention, surveillance,

and treatment. Recent developments in therapy are described below.

Myoclonus epilepsy with ragged red fiber (MERRF) is a multisystem

disorder characterized by myoclonus, seizures, ataxia, and myopathy

with ragged red fibers. Hearing loss, exercise intolerance, neuropathy,

ataxia, cervical lipomas, and short stature are often present. Ataxia and

lipomas can be a feature in adults or adult-onset MERRF. Cerebrospinal fluid (CSF) analysis reveals an elevated protein content. Almost all

MERRF patients have a mutation in the mtDNA tRNAlys gene, and the

m.8344A>G mutation in the mtDNA gene encoding the lysine amino

acid tRNA is responsible for 80–90% of MERRF cases.

Neuropathy, ataxia, and retinitis pigmentosa (NARP) is characterized by moderate diffuse cerebral and cerebellar atrophy and symmetric lesions of the basal ganglia on MRI (Figs. 468-7 and 468-8). A

heteroplasmic m.8993T>G mutation in the ATPase 6 subunit gene has

been identified as causative, which underscores the lack of definitive

genotype-phenotype correlation in mitochondrial diseases. Ragged

red fibers are not observed in muscle biopsy. When >95% of mtDNA

TABLE 468-3 Mitochondrial Diseases Due to Mitochondrial DNA (mtDNA) Point Mutations and Large-Scale Rearrangements

DISEASE PHENOTYPE

MOST FREQUENT mtDNA

MUTATIONS

HETEROPLASMIC/

HOMOPLASMIC MATERNAL

NARP, Leigh’s syndrome Loss of central vision leading to blindness in young

adult life

m.1778G>A, m.14484T>C,

m.3460G>A

Heteroplasmic Maternal

MELAS Mitochondrial encephalomyopathy, lactic acidosis,

and stroke-like episodes; may manifest only as

diabetes mellitus

Point mutation in tRNAleu Heteroplasmic Maternal

MERRF Myoclonic epilepsy, ragged red fibers in muscle,

ataxia, increased CSF protein, sensorineural deafness,

dementia

Point mutation in tRNAlys Heteroplasmic Maternal

Deafness Progressive sensorineural deafness, often induced by

aminoglycoside antibiotics

m.1555A>G mutation in 12S

rRNA

Homoplasmic Maternal

Nonsyndromic sensorineural deafness m.7445A>G mutation in 12S

rRNA

Homoplasmic Maternal

Chronic progressive

external

ophthalmoplegia (PEO)

Late-onset bilateral ptosis and ophthalmoplegia,

proximal muscle weakness, and exercise intolerance

Single deletions or duplications Heteroplasmic Mostly sporadic, somatic

mutations

Pearson’s syndrome Pancreatic insufficiency, pancytopenia, lactic acidosis Large deletion Heteroplasmic Sporadic, somatic mutations

Kearns-Sayre syndrome

(KSS)

External ophthalmoplegia, heart block, retinal

pigmentation, ataxia

The 5-kb “common deletion” Heteroplasmic Sporadic, somatic mutations

Abbreviations: CSF, cerebrospinal fluid; NARP, neuropathy, ataxia, and retinitis pigmentosa.

FIGURE 468-6 A 15-year-old girl with MELAS (mitochondrial encephalomyopathy,

lactic acidosis, and stroke-like episodes) due to m.A3243G (tRNALeu(UUR)), 85%

mutant heteroplasmy, presenting at age 5 with focal motor seizures, ataxia, and

short stature, with episodes of acute language and motor dysfunction and progress

cognitive impairment. The fluid-attenuated inversion recovery (FLAIR) MRI shows

increased signal intensity (white arrows) in the left temporal-parietal region in

addition to global mild volume loss (increased extra-axial cerebrospinal fluid

spaces).

Mitochondrial DNA and Heritable Traits and Diseases

3675CHAPTER 468

FIGURE 468-7 A 9-year-old girl with Leigh’s syndrome due to m.T8993G (ATPase

subunit 6), 99% heteroplasmy, presenting at age 14 months with a motor delay and

who underwent an MRI at 24 months, at which time she had just begun to walk. She

has moderate cognitive impairment, arm chorea, and distal leg dystonia. The fluidattenuated inversion recover (FLAIR) MRI shows symmetric bilateral increased

signal in the caudate nuclei (thin arrow) and putamen (thick arrow); only left-sided

lesions indicated with arrows.

FIGURE 468-8 A 12-year-old boy with Leigh’s syndrome due to m.T10191C (ND3

gene, complex I), heteroplasmy percentage not determined, presenting with

infantile spasms at 8 months of life. He responded well to adrenocorticotropic

hormone (ACTH), and his MRI and development were normal until 30 months when

he developed dystonia and progressive medically intractable epilepsy. The fluidattenuated inversion recover (FLAIR) MRI at 6 years of life shows global atrophy

with large extra-axial cerebrospinal fluid spaces, increased signal intensity in the

cortex (thin arrows), necrotic bilaterally symmetric lesions in the putamina, and

enlarged lateral ventricles due to loss of bilateral caudate nuclei volume (stars).

molecules are mutant, a more severe clinical, neuroradiologic, and neuropathologic picture (Leigh’s syndrome) emerges. Not uncommonly,

an infant is diagnosed with Leigh’s syndrome due to the m.8993T>G

mutation and not until several years later will the mother present with

symptoms of NARP, a situation that highlights the concept of a higher

threshold for lower levels of tissue heteroplasmy.

Point mutations in the mtDNA gene encoding the 12S rRNA

(m.A1555G) result in heritable nonsyndromic hearing loss. One such

mutation causes heritable ototoxic susceptibility to standard dosing of

aminoglycoside antibiotics, which opens a pathway for a simple pharmacogenetic test in the appropriate clinical settings. This is an example

of an ecogenetic disorder in that most people with this mutation do not

develop any symptoms until exposed to an external agent.

KSS, sporadic progressive external ophthalmoplegia (PEO), and

Pearson’s syndrome are three disease phenotypes caused by large-scale

mtDNA rearrangements including partial deletions or partial duplication. The majority of single large-scale rearrangements of mtDNA are

thought to result from clonal amplification of a single sporadic mutational event, occurring in the maternal oocyte during early embryonic

development. The typical mtDNA deletion specifically involves 4977

nucleotides, accounting for most KSS and PEO of mtDNA deletion

origin. Because germline involvement is rare, most cases are sporadic rather than inherited. KSS is characterized by the triad of onset

before age 20, chronic PEO, and pigmentary retinopathy. Cerebellar

syndrome, heart block, increased CSF protein content, diabetes mellitus, and short stature are also part of the syndrome. Single deletions/

duplication can also result in milder phenotypes such as PEO, characterized by late-onset PEO, proximal myopathy, and exercise intolerance. In

both KSS and PEO, diabetes mellitus and hearing loss are frequent accompaniments. Pearson’s syndrome is also characterized by infantile onset of

a sideroblastic anemia accompanied by lactic acidosis and failure to thrive

caused in part by exocrine pancreatic insufficiency. If the child survives,

the manifestations appear phenotypically similar to those of severe KSS

with myopathy, PEO, encephalopathy, and cardiomyopathy. Pearson’s

syndrome is generally caused by large-scale sporadic deletion of several

mtDNA genes that differ from the common deletion seen in KSS. Typically, the deletion size is larger in Pearson’s syndrome, and located with

different break points, than in KSS or PEO, but this is not always the case.

Two important dilemmas in classic mtDNA disease have benefited

from recent important research insights. The first relates to the greater

involvement of neuronal, muscular, renal, hepatic, and pancreatic manifestations in mtDNA disease in these syndromes. This observation

has appropriately been mostly attributed to the high energy utilization

of the involved tissues and organ systems and, hence, greater dependency on mitochondrial ETC integrity and health. However, because

mutations are stochastic events, mitochondrial mutations should

occur in any organ during embryogenesis and development. Recently,

additional explanations have been suggested based on studies of the

common m.3243A>G transition. The proportion of this mutation in

peripheral blood cells was shown to decrease exponentially with age. A

selective process acting at the stem cell level with a strong bias against

the mutated form would have its greatest effect to reduce the mutant

mtDNA only in highly proliferating cells, such as those derived from

the hematopoietic system. Tissues and organs carrying pathogenic

mtDNA mutations and having a lower cell turnover, such as brain,

nerve, or retina, would not benefit from this effect and, thus, would

accumulate mutational load and be the most affected.

The other dilemma arises from the observation that only a subset of mtDNA mutations accounts for the majority of the familial

mtDNA diseases. The random occurrence of mutations in the mtDNA

sequence should yield a more uniform distribution of disease-causing

mutations. However, recent studies using the introduction of one

severe and one mild point mutation into the female germline of experimental animals demonstrated selective elimination during oogenesis

of the severe mutation and selective retention of the milder mutation,

with the emergence of mitochondrial disease in offspring after multiple

generations. Thus, oogenesis itself can act as an “evolutionary” filter for

the most harmful mtDNA disease.

■ THE INVESTIGATION OF SUSPECTED mtDNA

DISEASE

The clinical presentations of classic syndromes, groupings of disease

manifestations in multiple organ systems, or unexplained isolated

3676 PART 16 Genes, the Environment, and Disease

presentations of one of the disease features of a classic mtDNA syndrome should prompt a systematic clinical investigation as outlined in

Fig. 468-9. Indeed, mitochondrial disease should be considered in the

differential diagnosis of any progressive multisystem disorder. Despite

the centrality of disruptive oxidative phosphorylation, an elevated

blood lactate level is neither specific nor sensitive, because there are

many causes of blood lactic acidosis and many patients with mtDNA

defects presenting at any age may have normal blood lactate levels. An

elevated CSF lactate is a more specific test for mitochondrial disease if

there is central nervous system involvement. The serum creatine kinase

may be elevated but is often normal, even in the presence of a proximal

myopathy. Recently, testing for elevated levels of growth differentiating

factor 15 (GDF15) has shown a high degree of sensitivity and specificity in those with a mitochondrial myopathy, but the degree of elevation

for an individual patient reflects the severity of the illness and does

not seem to be a sensitive marker of disease activity. Urinary organic

acids (specifically TCA cycle intermediates) and amino acids (alanine,

proline) may also be abnormal, reflecting metabolic as well as kidney

proximal tubule dysfunction. Every patient with seizures, episodes of

confusion or atypical behavioral changes, or cognitive decline should

have an electroencephalogram. A brain computed tomography (CT)

scan may show calcified basal ganglia or bilateral hypodense regions

with cortical atrophy. MRI is indicated in patients with brainstem signs

or stroke-like episodes.

For some mitochondrial diseases, it is possible to obtain an accurate

diagnosis with a simple molecular genetic screen. For examples, 95%

of patients with LHON harbor one of the three mtDNA point mutations (m.11778A>G, m.A3460A>G, or m.14484T>C). These patients

have very high levels of mutated mtDNA in peripheral blood cells, and

therefore, it is appropriate to send a blood sample for molecular genetic

analysis by polymerase chain reaction (PCR) or restriction fragment

length polymorphism (RFLP). The same is true for most MERRF

patients who harbor a point mutation in the lysine tRNA gene at position 8344. In contrast, patients with the m.3243A>G MELAS mutation

often have low levels of mutated mtDNA in blood. If clinical suspicion

is strong enough to warrant peripheral blood testing, then patients with

a negative result should have testing repeated using a saliva sample

or be investigated further by performing a skeletal muscle biopsy to

obtain mtDNA from a relatively nonreplicative tissue.

Muscle biopsy histochemical analysis had been the historical cornerstone for investigation of patients with suspected mitochondrial

disease. Histochemical analysis may show subsarcolemmal accumulation of mitochondria with the appearance of ragged red fibers, especially in those with mtDNA mutations affecting the tRNA and rRNA

genes. Electron microscopy might show abnormal mitochondria with

paracrystalline inclusions. Muscle histochemistry may show COXdeficient fibers, which indicate mitochondrial dysfunction (Fig. 468-5).

Respiratory chain complex assays may also show reduced enzyme

function. If enzymatic or polarographic data are used to aid in the

confirmation of diagnosis, a standard method of analysis should be

employed. Either of these two abnormalities, within the exact context

of established peer-reviewed criteria, may confirm the presence of a

mitochondrial disease, to be followed by an in-depth molecular genetic

analysis. In most major centers, genetic testing has become the primary

means of obtaining a definitive diagnosis, using muscle pathology

and biochemistry to assist with interpretation of inconclusive genetic

results.

Recent evidence has provided important insights into the importance of nuclear-mtDNA genomic cross-talk and has provided a

descriptive framework for classifying and understanding disorders that

emanate from perturbations in this cross-talk. Although not strictly

considered as mtDNA genetic disorders, manifestations do overlap

those highlighted above (Fig. 468-10).

IMPACT OF HOMOPLASMIC SEQUENCE

VARIATION ON HERITABLE TRAITS AND

DISEASE

The relationship among the degree of heteroplasmy, tissue distribution

of the mutant mtDNA, and disease phenotype simplifies inference of

a clear causative relationship between heteroplasmic mutation and

disease. With the exception of certain mutations (e.g., those causing

most cases of LHON), drift to homoplasmy of such mutations would be

precluded normally by the severity of impaired oxidative phosphorylation and the consequent reduction in reproductive fitness. Therefore,

sequence variants that have reached homoplasmy should be neutral in

terms of human evolution and, hence, useful only for tracing human

evolution, demography, and migration, as described above. Thus, novel

homoplasmic variants are seldom pathogenic. One important exception is in the case of one or more of the homoplasmic population-level

variants, which designate the mtDNA haplogroup J, and the interaction with the mtDNA mutations causing LHON. Reduced disease

predilection suggests that one or more of the ancient sequence variants

designating mtDNA haplogroup J appear to attenuate predisposition to

degenerative disease, in the presence of other risk factors. Whether or

not additional epistatic interactions between population-level mtDNA

haplotypes and common health conditions will be found remains to

be determined. If such influences do exist, then they are more likely

to be relevant to health conditions in the postreproductive age groups,

wherein evolutionary filters would not have had the opportunity to

censor deleterious effects and interactions and wherein the effects of

oxidative stress during aging or with poor diet or lack of exercise may

play a role. Although much has been written about the possible associations between population-level common mtDNA variants and human

health and disease phenotypes or adaptation to different environmental influences (e.g., climate), a word of caution is in order.

Many studies that purport to show such associations with phenotypes such as longevity, athletic performance, and metabolic and

neurodegenerative disease are limited by small sample sizes, possible

genotyping inaccuracies, and the possibility of population stratification

or ethnic ancestry bias. Because mtDNA haplogroups are so prominently partitioned along phylogeographic lines, it is difficult to exclude

the possibility that a haplogroup for which an association has been

reported is simply a marker for otherwise unappreciated population

Clinical investigations

Blood: creatine kinase, liver functions,

glucose, lactate

Urine: organic and amino acids

CSF: glucose, protein, lactate

Cardiac x-ray, ECG, ECHO

EEG, EMG, nerve conduction

Brain CT/MRI

PCR/RFLP analysis of

blood for known mutations

Histochemistry Study of respiratorychain complexes

activities

Yes

No

Molecular genetic analysis

rearrangements

PCR/RFLP for common point mutation

mtDNA automated sequencing

Specific point mutation syndrome:

e.g., MELAS, MERRF, and LHON

Muscle biopsy

FIGURE 468-9 Clinical and laboratory investigation of a suspected mitochondrial

DNA (mtDNA) disorder. CSF, cerebrospinal fluid; CT, computed tomography; ECG,

electrocardiogram; ECHO, echocardiography; EEG, electroencephalogram; EMG,

electromyogram; LHON, Leber’s hereditary optic neuropathy; MELAS, mitochondrial

encephalomyopathy, lactic acidosis, and stoke-like episodes; MERRF, myoclonic

epilepsy with ragged red fibers; MRI, magnetic resonance imaging; PCR, polymerase

chain reaction; RFLP, restriction fragment length polymorphism.

Mitochondrial DNA and Heritable Traits and Diseases

3677CHAPTER 468

Multiple ∆mtDNA

dNTP pool

Pyrimidine salvage

mtDNA depletion

adPEO

Twinkle

Twinkle

Alpers’ like

IOSCA

Alpers’s.

SCAE

Pol γA

Pol γ

adPEO

arPEO

A

B

ANT1

adPEO

Deoxyguanosine kinase

MPV17

Thymidine kinase (TK2)

RRMB2 (p53-R2)

Succinyl-CoA synthase

(SUCLA2, SUCLG1)

TP

Thymidine phosphorylase

Patient Control

FIGURE 468-10 Disorders associated with perturbations in nuclear-mitochondrial genomic crosstalk. Clinical features and genes associated with multiple mitochondrial DNA (mtDNA) deletions,

mtDNA depletion, and mitochondrial neurogastrointestinal encephalomyopathy syndromes.

ANT, adenine nucleotide translocators; adPEO, autosomal dominant progressive external

ophthalmoplegia; arPEO, autosomal recessive progressive external ophthalmoplegia; IOSCA,

infantile-onset spinocerebellar ataxia; SCAE, spinocerebellar ataxia and epilepsy. (Reproduced with

permission from A Spinazzola, M Zeviani: Disorders from perturbations of nuclear-mitochondrial

intergenomic cross-talk. J Intern Med 265:174, 2009.)

tissue is expected to be associated with mitochondrial

dysfunction, as reflected in an age-associated patchy

and reduced COX activity on histochemical staining,

especially in skeletal and cardiac muscle and brain.

A particularly well-studied and potentially important

example is the accumulation of mtDNA deletions and

COX deficiency observed in neurons of the substantia

nigra in Parkinson’s disease patients.

The progressive accumulation of ROS has been

proposed as the key factor connecting mtDNA mutations with aging and age-related disease pathogenesis

(Fig. 468-11). As noted above, ROS are a by-product

of normal oxidative phosphorylation and are removed

by detoxifying antioxidants into less harmful moieties;

however, environmental factors or mutations that result

in exaggerated production of ROS or impaired removal

result in ROS accumulation and subsequent cellular

injury. One of the main targets for ROS-mediated injury is

DNA, and mtDNA is particularly vulnerable because of its

proximity to the origin of free radical production, the lack

of protective histones, and less efficient injury repair systems compared with nuclear DNA. In turn, accumulation

of mtDNA mutations results in inefficient oxidative phosphorylation, with the potential for excessive production of

ROS, generating a “vicious cycle” of cumulative mtDNA

damage. Indeed, measurement of the oxidative stress

biomarker 8-hydroxy-2-deoxyguanosine has been used

to measure age-dependent increases in mtDNA oxidative

damage at a rate exceeding that of nuclear DNA. It should

be noted that mtDNA mutations can potentially occur in postmitotic

cells as well, because mtDNA replication is not synchronized with the

cell cycle. Two other proposed links between mtDNA mutation and

aging, besides ROS-mediated tissue injury, are the perturbations in

efficiency of oxidative phosphorylation with disturbed cellular aerobic

function and perturbations in apoptotic pathways, whose execution

steps involve mitochondrial activity.

heterogeneity, wherein a nongenetic (societal or environmental) difference among the populations marked by the mtDNA haplogroup

differences is actually causally related to the disease of interest. The

experimental difficulty in generating cellular or animal models to test

the functional influence of homoplasmic sequence variants (as a result of

mtDNA polyploidy) further compounds the challenge. The most likely

formulation is that the risk conferred by different mtDNA haplogroupdefining homoplasmic mutations for common diseases depends on the

concomitant nuclear genomic background, together with environmental influences. Progress in minimizing potentially misleading associations in mtDNA heritable trait and disease studies should include

ensuring adequate sample size taken from a large sample recruitment

base, using carefully matched controls and population structure determination, and performing analysis that takes into account epistatic

interactions with other genomic loci and environmental factors.

IMPACT OF ACQUIRED SOMATIC mtDNA

MUTATION ON HUMAN HEALTH AND

DISEASE

Studies on aging humans and animals have shown a potentially

important correlation of age with the accumulation of heterogeneous

mtDNA mutations, especially in organ systems that undergo the most

prominent age-related degenerative tissue phenotype. Sequencing of

PCR-amplified single mtDNA molecules has demonstrated an average

of two to three point mutations per molecule in elderly subjects when

compared with younger ones. Point mutations observed include those

responsible for known heritable heteroplasmic mtDNA disorders, such

as the m.3344A>G and m.3243A>G mutations responsible for the

MERRF and MELAS syndromes, respectively. However, the cumulative burden of these acquired somatic point mutations with age was

observed to remain well below the threshold expected for phenotypic

expression (<2%). Point mutations at other sites not normally involved

in inherited mtDNA disorders have also been shown to accumulate

to much higher levels in some tissues of elderly individuals, with

the description of tissue-specific “hot spots” for acquired somatic

mtDNA point mutations. Likewise, an age-associated and tissuespecific accumulation of mtDNA deletions has been observed, including deletions involved in known heritable mtDNA disorders, as well as

others. The accumulation of functional mtDNA deletions in a given

Mutant

mitochondrial

proteins

Damaged

mitochondrial

proteins

Error-prone

DNA Pol-γ

Nuclear

DNA damage

ROS

Decreased

DNA repair

Apoptosis

DNA

mutations

O2

O2

H2O2

H2O OH

Aging

X − X

FIGURE 468-11 Multiple pathways of mitochondrial DNA (mtDNA) damage and

aging. Multiple factors may impinge on the integrity of mitochondria that lead to

loss of cell function, apoptosis, and aging. The classic pathway is indicated with

blue arrows; the generation of reactive oxygen species (ROS; superoxide anion,

hydrogen peroxide, and hydroxyl radicals), as a by-product of mitochondrial

oxidative phosphorylation, results in damage to mitochondrial macromolecules,

including the mtDNA, with the latter leading to deleterious mutations. When these

factors damage the mitochondrial energy-generating apparatus beyond a functional

threshold, proteins are released from the mitochondria that activate the caspase

pathway, leading to apoptosis, cell death, and aging. (Reproduced with permission

from L Loeb et al: The mitochondrial theory of aging and its relationship to reactive

oxygen species damage and somatic mtDNA mutations. Proc Natl Acad Sci USA

102:18769, 2005. Copyright (2005) National Academy of Sciences, U.S.A.)

3678 PART 16 Genes, the Environment, and Disease

Genetic intervention studies in animal models have sought to

clarify the potential causative relationship between acquired somatic

mtDNA mutation and the aging phenotype and the role of ROS in

particular. Replication of the mitochondrial genome is mediated by

the activity of the nuclear-encoded POLG. A transgenic homozygous

mouse knock-in mutation of this gene renders the polymerase enzyme

deficient in proofreading and results in a threefold to fivefold increase

in mtDNA mutation rate. Such mice develop a premature aging phenotype, which includes subcutaneous lipoatrophy, alopecia, kyphonia, and weight loss with premature death. Although the finding of

increased mtDNA mutation and mitochondrial dysfunction with age

has been solidly established, the causative role and specific contribution of mitochondrial ROS to aging and age-related disease in humans

have yet to be proved. Similarly, although many tumors display higher

levels of heterogeneous mtDNA mutations, a causal relationship to

tumorigenesis has not been proved.

Besides the age-dependent acquired accumulation in somatic cells

of heterogeneous point mutations and deletions, a quite different effect

of nonheritable and acquired mtDNA mutations has been described

affecting tissue stem cells. In particular, disease phenotypes attributed

to acquired mtDNA mutation have been observed in sporadic and

apparently nonfamilial cases involving a single individual or even tissue,

usually skeletal muscle. The presentation consists of decreased exercise

tolerance and myalgias, sometimes progressing to rhabdomyolysis. As

in the case of the sporadic, heteroplasmic, large-scale deletion, classic

syndromes of chronic PEO, Pearson’s syndrome, and KSS, the absence of

a maternal inheritance pattern and the finding of limited tissue distribution suggest a molecular pathogenic mechanism emanating from mutations arising de novo in muscle stem cells after germline differentiation

(somatic mutations that are not sporadic and occur in tissue-specific

stem cells during fetal development or in the postnatal maintenance or

postinjury repair stage). Such mutations would be expected to be propagated only within the progeny of that stem cell and affect a singular

tissue within a given individual, without evidence of heritability.

PROSPECTS FOR CLINICAL MANAGEMENT

OF mtDNA DISEASE

■ TREATMENT OF mtDNA DISORDERS

No specific curative treatment for mtDNA disorders is currently available; therefore, the management of mitochondrial disease is largely supportive. Management issues may include early diagnosis and medical

management of epilepsy, gastrointestinal dysfunction, weakness, diabetes mellitus, cardiac dysrhythmia, hearing loss, endocrinopathy, ptosis,

and cataracts. The value of aggressive symptom management cannot be

understated. Less specific interventions in the case of other disorders

involve combined treatment strategies including dietary intervention

and removal of toxic metabolites. Cofactors and vitamin supplements

are widely used in the treatment of diseases of mitochondrial oxidative

phosphorylation, although there is little evidence, apart from anecdotal

reports, to support their use. This includes administration of artificial

electron acceptors, including vitamin K3

, vitamin C, and ubiquinone

(CoQ10); administration of cofactors (coenzymes) including riboflavin,

carnitine, and creatine; and use of oxygen radical scavengers, such as

vitamin E, copper, selenium, ubiquinone, and idebenone. Drugs that

could interfere with mitochondrial function, such as the anesthetic

agent propofol, barbiturates, and high doses of valproate, should be

avoided. The use of valproate in patients with pathogenic mutations in

POLG and possibly other mutations affecting mtDNA stability and replication is especially contraindicated. Supplementation with the nitric

oxide synthase substrate l-arginine and, more recently, l-citrulline has

been advocated as a vasodilator treatment during stroke-like episodes

as well as for chronic management in patients with MELAS. Open-label

studies demonstrate that levoarginine and levocitrulline may be helpful

in reducing the stroke-like symptoms in MELAS but may have serious

side effects. As CSF folate deficiency has been reported in some cases of

mitochondrial disease, this can be treated with folinic acid.

The physician should also be familiar with environmental interactions, such as the strong and consistent association between visual loss

in LHON and smoking or ethanol consumption. A clinical penetrance

of 93% was found in men who smoked. Asymptomatic carriers of an

LHON mtDNA mutation should, therefore, be strongly advised not

to smoke and to moderate their alcohol intake. Although not a cure,

these interventions might stave off the devastating clinical manifestations of the LHON mutation. Another example is strict avoidance of

aminoglycosides in the familial syndrome of ototoxic susceptibility to

aminoglycosides in the presence of the mtDNA m.1555A>G mutation

of the 12SrRNA encoding gene.

Clinical trials using novel agents have been initiated and launched.

These agents include α-tocotrienol (PTC-743 previously termed EPI743, PTC Therapeutics), REN-001 (Reneo Pharmaceuticals), ASP0367

(Astellas Pharmaceuticals), and elamipretide (Stealth Biotherapeutics).

In an open-label study of α-tocotrienol used to treat 10 children with

Leigh’s syndrome, there were improvements in the primary endpoints,

including the Newcastle Pediatric Mitochondrial Diseases Scale, the

Gross Motor Function Measure, and the PedsQL Neuromuscular

Module. Ongoing studies continue for α-tocotrienol in children with

epilepsy, along with REN001, ASP0367, and elamipretide in adults

with primary mitochondrial myopathy. Conclusive progress has experienced some delay due to the COVID-19 pandemic.

GENETIC COUNSELING, PRENATAL

DIAGNOSIS, AND PGD IN mtDNA

DISORDERS

The provision of accurate genetic counseling and reproductive options

to families with mtDNA mutations is challenging due to the unique

genetic features of mtDNA inheritance that distinguish it from Mendelian genetics. mtDNA defects are transmitted by maternal inheritance.

mtDNA de novo mutations are often large deletions, affect one family

member, and usually represent no significant risk to other members of

the family. In contrast, mtDNA point mutations or duplications can be

transmitted maternally. Accordingly, the father of an affected individual has no risk of harboring the disease-causing mutation, and a male

cannot transmit the mtDNA mutation to his offspring. In contrast, the

mother of an affected individual usually harbors the same mutation but

may be completely asymptomatic. This wide phenotypic variability is

primarily related to the phenomena of heteroplasmy and the mutation

load carried by different members of the same family. Consequently,

a symptomatic or asymptomatic female harboring a disease-causing

mutation in a heteroplasmic state will transmit to her offspring variable amounts of the mutant mtDNA molecules. The offspring will be

symptomatic or asymptomatic primarily according to the mutant load

transmitted via the oocyte and, to some extent, subsequent mitotic

segregation during development. Interactions with the mtDNA haplotype background or nuclear human genome (as in the case of LHON)

serve as an additional important determinant of disease penetrance.

Because the severity of the disease phenotype associated with the

heteroplasmic mutation load is a function of the stochastic differential

segregation and copy number of mutant mtDNA during the oogenesis

bottleneck and, subsequently, following tissue and organ development

in the offspring, it is rarely predictable with any degree of accuracy.

For this reason, prenatal diagnosis (PND) and PGD techniques that

have evolved into integral and well-accepted standards of practice are

severely hampered in the case of mtDNA-related diseases.

The value of PND and PGD is limited, partly due to the absence

of data on the rules that govern the segregation of wild-type and

mutant mtDNA species (heteroplasmy) among tissue in the developing

embryo. Three factors are required to ensure the reliability of PND and

PGD: (1) a close correlation between the mutant load and the disease

severity, (2) a uniform distribution of mutant load among tissues, and

(3) no major change in mutant load with time. These criteria are suggested to be fulfilled for the NARP m.8993T>G mutation but do not

seem to apply to other mtDNA disorders. In fact, the level of mutant

mtDNA in a chorionic villous or amniotic fluid sample may be very

different from the level in the fetus, and it would be difficult to deduce

whether the mutational load in the prenatal samples provides clinically

useful information regarding the postnatal and adult state.

Mitochondrial DNA and Heritable Traits and Diseases

3679CHAPTER 468

■ PREVENTION OF MITOCHONDRIAL DISEASE

INHERITANCE BY ASSISTED REPRODUCTIVE

TECHNOLOGIES

Because the treatment options for patients with mitochondrial disease

are rather limited, with no current U.S. Food and Drug Administration (FDA)–approved therapies for established mitochondrial DNA

disease, preventive interventions that eliminate the likelihood of

transmission of affected mtDNA into offspring are desirable. The poor

reliability of prenatal and preimplantation approaches in predicting

mitochondrial DNA disease has resulted in the search for alternative

preventive approaches. The common purpose underlying various

emerging approaches is to reduce mutant heteroplasmy levels to a

level below a pathogenic threshold. This is based on the observed

relationship between heteroplasmy and disease inheritance patterns,

which indicates that even a small increase in copy number of nonmutant mtDNA molecules in the fertilized egg can exceed the threshold

required to ameliorate serious clinical disease. Use of gene editing, with

clustered regularly interspaced short palindromic repeats (CRISPR) or

mitochondrial-targeted TALEN (transcription activator-like effector

nucleases) technology, for example, to shift the heteroplasmy load

in affected tissues will require future development of corrective gene

delivery techniques. Likewise, induced pluripotent cell technology has

not yet met with widespread success in the preclinical research setting.

This has prompted the application of mitochondrial replacement therapy (MRT) approaches (Fig. 468-12). These approaches substitute in

vitro the entire oocyte or zygote complement of mitochondria, together

FIGURE 468-12 Mitochondrial replacement techniques—maternal spindle transfer and pronuclear transfer. In both procedures, some mutant mtDNA, estimated at 1–2%,

might be carried over together with the spindle or pronucleus, but the levels are low enough to avoid disease risk. IVF, in vitro fertilization. (From MJ Falk et al: Mitochondrial

replacement techniques—Implications for the clinical community. N Engl J Med 374:1103, 2016. Copyright © 2016, Massachusetts Medical Society. Reprinted with

permission from Massachusetts Medical Society.)

3680 PART 16 Genes, the Environment, and Disease

■ DEFINITION

In telomere diseases (also called telomeropathies or telomere spectrum

disorders), organ dysfunction is caused by loss of the ends of chromosomes, a process termed accelerated telomere attrition. Inadequate

repair or insufficient protection of telomeres and their resulting erosion induces cell death, deficient cell proliferation, and chromosome

instability; affected tissues show defective organ regeneration, fibrosis

469 Telomere Disease

Rodrigo T. Calado, Neal S. Young

with their mtDNA from the carrier mother, with the unaffected complement of mitochondria and their unaffected mtDNA from a donor

woman. This can be accomplished either by removing and transferring

the carrier mother’s spindle with her nuclear DNA into the unfertilized

oocyte of the donor or, alternatively, by transferring the pronucleus

from the fertilized oocyte of the carrier mother to the unfertilized

donor oocyte from which the pronucleus has been removed. These

approaches provide a “bulk” substitution and hence do not target the

specific mtDNA mutation, and they are potentially applicable to a wide

variety of mtDNA disorders. This is a form of germline genetic therapy,

and therefore, it projects onto future generations in the case of a female

offspring. Accordingly, ethical and regulatory bodies have appropriately weighed in on the societal implications of such approaches and

have been tentatively supportive of human clinical investigation for

situations that would prevent great suffering and when the clinical

need is clear and unambiguous, subject to specified conditions and

principles and subject to ethical scrutiny. Several such studies have

been initiated, and careful examination and follow-up are needed to

determine developmental and longer-term health and fertility of children who have undergone genetic manipulation at the earliest stages of

human development and whose genomes comprise separate maternal

origins of nuclear and mtDNA genomes. It has been recommended

that such studies be limited to male offspring, who cannot then transmit the donor mtDNA to future generations, until such time as the

health, ethical, and societal issues are well understood and live up to

the exciting promise of reducing the burden of clinical mtDNA disease

in the future.

■ FURTHER READING

Alston CL et al: The genetics and pathology of mitochondrial disease.

J Pathol 241:236, 2017.

Camp K et al: Nutritional interventions in primary mitochondrial

disorders: Developing an evidence base. Mol Genet Metab 119:187,

2016.

DiMauro S: Mitochondrial encephalomyopathies—Fifty years on: The

Robert Wartenberg Lecture. Neurology 81:281, 2013.

DiMauro S et al: The clinical maze of mitochondrial neurology. Nat

Rev Neurol 9:429, 2013.

Haas RH et al: Mitochondrial disease: A practical approach for primary care physicians. Pediatrics 129:1326, 2007.

Koopman WJ et al: Monogenic mitochondrial disorders. N Engl J Med

366:1132, 2012.

Parikh S et al: Patient care standards for primary mitochondrial

disease: A consensus statement from the Mitochondrial Medicine

Society. Genet Med 19:1380, 2017.

Russell OM et al: Mitochondrial diseases: Hope for the future. Cell

181:168, 2020.

Saneto RP et al (eds): Mitochondrial Case Studies, Underlying Mechanisms and Diagnosis. London, Academic Press/Elsevier, 2016.

or replacement by fat, and a proclivity for cancer. A variety of regenerative disorders affecting especially the bone marrow, lungs, liver, and

skin share telomere dysfunction and accelerated loss as their common

molecular mechanism. Cross-sectional data of average telomere length

in cohorts of different ages and longitudinal studies in individuals indicate that telomeres shorten with aging at an average of 50 base pairs/

year in peripheral blood leukocytes. Despite shortening of telomeres

over time, normal aging is not associated with the development of a

telomere disease from short telomeres. In normal aging, sufficient stem

cell number and function are maintained to sustain vital processes.

For example, in stem cell transplant, even a limited number of donor

hematopoietic cells maintains normal hematopoiesis for many years, at

least in part related to normal telomerase function and telomere repair.

Telomere length associates with life span in the general population.

While shorter leukocyte telomeres correlate with increased mortality

risk, especially from nonmalignant causes, telomere loss is not established as the cause of physiologic aging.

■ DISEASE MECHANISM

Telomeres, the physical termini of linear chromosomes, are repeated

hexanucleotide sequences physically associated with specific proteins.

Telomeres function to protect the chromosome ends against recognition as damaged or infectious DNA by the DNA repair machinery

(Fig. 469-1). During mitosis, the DNA polymerase employs an RNA

oligonucleotide with a 3′ hydroxyl group to prime replication. The

primer dissociates as the DNA polymerase advances along the template strand, and a gap is left at the ends of linear DNA molecules: the

newly synthesized DNA strand is necessarily shorter than the original

template—the “end-replication problem.” Chromosome erosion is thus

inevitable with mitotic cell division, but the noncoding telomeric long,

repetitive structure buffers loss of genetic information. In human cells,

telomeres are composed of hundreds to thousands of TTAGGG tandem

repeats in the leading and CCCTAA in the lagging DNA strand. At

birth, telomeres are relatively long, but they inexorably shorten with

chronological aging (Fig. 469-1). In an individual cell, critically short

telomere length triggers the p53 pathway, usually leading to proliferative

arrest, senescence, and apoptosis. Telomere loss is the molecular basis

for the “Hayflick phenomenon,” the limit to cell division and thus to cell

proliferation in tissue culture. If a cell overcomes proliferation arrest,

extremely short telomeres engage the DNA damage repair machinery,

and chromosome end-to-end fusions, chromosome breaks, aneuploidy,

and chromosome instability may occur. In addition to the telomere

repeated sequences, a group of specialized proteins, collectively termed

shelterins, directly bind to or indirectly associate with telomeres, assisting in the organization of the telomere tertiary structure and inhibiting

activity of DNA damage response proteins (Fig. 469-1).

To escape telomere attrition, cells with high proliferative demand,

including embryonic and adult stem cells, lymphocytes, and the

majority of cancer cells have at least two mechanisms to preserve

telomere length: homologous recombination (important in malignant cell transformation) and the synthesis of telomeric repeats (in

physiologic telomere maintenance). The telomerase machinery adds

GTTAGG hexanucleotides to the 5′ end of the leading DNA strand

using telomerase, a reverse transcriptase enzyme (TERT), and TERC,

its RNA template (Fig. 469-1). The telomerase holoenzyme complex is

composed of two copies of TERT, TERC, and dyskerin, and associated

proteins. TERC binds to TERT and serves as the RNA template for its

function as a reverse transcriptase. Dyskerin, encoded by DKC1, stabilizes the complex, and TCAB1, encoded by the WRAP53 gene, aids

telomerase trafficking to the Cajal bodies, nuclear structures for ribonucleoprotein processing where telomerase associates with telomeres

for elongation. Telomerase expression is tightly regulated. MYC, sex

hormones, and many additional factors stimulate TERT transcription.

In mature cells, the TERT gene is repressed. In addition, shelterin proteins can also regulate telomerase function and processivity, modulating its catalytic activity on telomeres. Other proteins also are important

for telomere length maintenance. RTEL1, an essential DNA helicase,

dismantles T-loops and resolves G-quadruplexes, ensuring adequate

telomere elongation.

Telomere Disease

3681CHAPTER 469

When telomeres are critically short, the DNA damage response machinery may be recruited, mistaking

telomeres for damaged or infectious DNA and forcing

inappropriate repair. Activation of this pathway may

cause chromosome instability due to end-to-end fusion

of chromosomes or translocations; these alterations

generate genomic instability and potentially malignant

clones of cells. That telomere dysfunction increases the

risk of cancer development has been demonstrated in

murine models of telomerase deficiency, and patients

with telomere diseases are prone to develop acute

myeloid leukemia and head and neck squamous cell

carcinomas.

■ GENETICS

The pattern of inheritance is variable: X-linked, autosomal recessive, and autosomal dominant. Complex

genetic architecture also occurs, and affected patients

may inherit pathogenic loss-of-function variants in

more than one gene involved in the same telomere biology pathway. At least 15 genes have been implicated in

the etiology of telomeropathies (Table 469-1).

■ CLINICAL MANIFESTATIONS

Presentation of telomere disease in the clinic is highly

variable—in the tissues affected, in the severity of organ

dysfunction, and in patterns of disease within families

and between families with similar mutations. In a

same family, one individual may be severely affected,

but close relatives carrying the same mutation may

be asymptomatic and with normal laboratory results.

Asymptomatic carriers, however, may have subclinical

organ dysfunction, which may be detected by directed

or specialized testing (reduced forced vital capacity on

pulmonary function test, hypocellular bone marrow

at biopsy, hepatic steatosis on ultrasound). Somatic

genetic rescue may occur in telomere diseases and

modulate the clinical phenotype. Rescue is a rare

spontaneous genetic event in a somatic cell, conferring

a selective advantage and annulling the effect of the

original pathogenic germline mutation. In telomeropathies, somatic genetic rescue has been detected in

hematopoietic cells, so that one clonal population

adequately produces blood cells and mitigates the marrow failure phenotype. Generally, relatives sharing the

same mutation and short telomeres may have different

organs affected: for instance, while one individual may

have aplastic anemia, another within the pedigree may

suffer from pulmonary fibrosis. Environmental regenerative stresses, factors such as smoking and alcohol consumption,

and viral infection may increase susceptibility to organ damage in

these patients and contribute to disease heterogeneity.

Disease anticipation, in which clinical phenotype manifests at an

earlier age in successive generations, is observed in some families with

telomeropathies, due to the direct inheritance of short telomeres present in sperm and oocytes.

The diagnosis of a telomere disease is suggested by personal and

family history, strengthened by simple measurement of leukocyte telomere length, and usually definitively established by next-generation

sequencing for genes encoding telomere repair enzyme complex and

shelterin components.

Dyskeratosis Congenita Dyskeratosis congenita is the classic

telomere disease, a mainly pediatric syndrome diagnosed in the first

two decades of life. Affected children often have at least two features

of the mucocutaneous triad of ungual dystrophy, reticular skin pigmentation, and oral leukoplakia (Fig. 469-2). In severe syndromes,

affected newborns have cerebellar hypoplasia (Hoyeraal-Hreidarsson

B Age (years)

A

0

Telomere length (kb)

2

4

6

8

10

12

14

Shelterin

TRF2

Rap1

TPP1

TIN2 TRF1

POT1

Dyskerin

AAUCCC

Telomerase

AATCCCAATCCCAATCCC-5'

GAR

NOP10

NHP2

RNA

template

Centromere

Chromosome

T loop

D loop

3'

5'

TERT

Average loss of 40–60 base pairs/year

0 20 40 60 80 100

TTAGGGTTAGGGTTAGGGTTAGGGTTAGGG-3'

FIGURE 469-1 Telomeres and telomerase. A. Telomeres are ribonucleoprotein structures located at

the termini of linear chromosomes inside the cell nucleus composed of hundreds of tandem hexameric

DNA repeats. A group of proteins bind directly or indirectly to telomere sequences in order to provide

protection to the structure and are collectively termed shelterin or telosome (TRF1, TRF2, TIN2, POT1,

TPP1, and RAP1). As the 3′ end of the leading strand forms a single-stranded overhang, it folds back and

invades the telomeric double helix, forming a lariat termed T-loop. The telomerase complex is composed

of the enzyme telomerase reverse transcriptase (TERT), its RNA component (TERC), the protein dyskerin,

and associated proteins (NHP2, NOP10, and GAR). This enzymatic complex elongates telomeres by

adding GTTAGG hexameric repeats to the 3′ end of the telomeric leading strand, using a sequence in

TERC as the template. B. The average telomere length in human leukocytes varies: it is longer at birth

(10–11 kb) and progressively shortens with aging (5–7 kb at age 90 years) at an average loss of 40–60

base pairs/year. However, there is significant variability in telomere length in each given age.

Pathologic accelerated telomere attrition has a genetic origin.

Germline loss-of-function mutations in genes involved in telomere

maintenance and function impair telomere length repair, thus increasing the rate of telomere erosion in highly proliferative cells. Telomeres

reach critically short lengths faster than with normal aging; the consequences are limited cell proliferation and impaired tissue regeneration.

Some organs appear to be particularly susceptible to telomere erosion.

Billions of blood cells are produced daily (Chap. 96), and telomere

attrition curtails cell proliferation, producing a hypocellular marrow

and often low blood counts. The liver also is an organ with high proliferative capacity, and telomere dysfunction impairs hepatic regeneration

after injury, with a variety of pathologic consequences. The lung alveolar epithelium is in contact with exogenous toxins that stimulate regeneration, and telomere loss may hinder these physiologic responses.

However, it remains unclear why other regenerative tissues, such as the

intestine, are less affected by telomere dysfunction; the mechanism by

which telomere loss provokes a fibrotic response in the lungs (pulmonary fibrosis), an adipose response in the marrow (aplastic anemia),

and both in the liver (hepatic steatosis and cirrhosis) is also unclear.

3682 PART 16 Genes, the Environment, and Disease

TABLE 469-1 Genetic Variants in 13 Genes Involved in Telomere Maintenance, Inheritance

Pattern, and Phenotype

GENE

DYSKERATOSIS

CONGENITA

APLASTIC

ANEMIA

PULMONARY

FIBROSIS CIRRHOSIS MDS/LEUKEMIA

Telomerase

DKC1 XL

TERT AD/AR AD/AR AD AD AD/AR

TERC AD/AR AD AD AD AD

NOP10 AR

NHP2 AR

WRAP53 AR

Shelterin

TINF2 AD AD AD

TERF2 AD

ACD AD

Others

RTEL1 AR AD/AR AD AD

CTC1 AR AR

PARN AD

USB1 AD

ZCCHC8 AD AD

NAF1 AD

Abbreviations: AD, autosomal dominant; AR, autosomal recessive; MDS, myelodysplastic syndrome; XL, X-linked.

FIGURE 469-2 Skin manifestations of dyskeratosis congenita. The pediatric syndrome dyskeratosis congenita

is characterized by the mucocutaneous triad of (A) reticular skin pigmentation, (B) oral leukoplakia, and (C, D) nail

dystrophy.

syndrome) or exudative retinopathy (Revesz syndrome) (Fig. 469-3).

Telomeres are usually extremely short, below the first percentile

expected for age (Fig. 469-4). Most patients with dyskeratosis congenita develop bone marrow failure, often requiring transfusions and

ultimately bone marrow transplant. Pulmonary fibrosis appears in as

many as 20% of cases and liver disease in 10%, often after bone marrow

transplant for hematopoietic failure. Other tissues and organs also may

be affected (Fig. 469-3). The most common mutations in dyskeratosis

congenita patients are in the DKC1, TINF2, TERT, TERC, and RTEL1

genes, and triallelic inheritance (involving two genes in the same pathway) also may occur (Table 469-1).

Bone Marrow Failure Aplastic anemia

(Chap. 102) is the most common major clinical

manifestation of dyskeratosis congenita. However, young or older patients carrying a telomere defect, without typical physical stigmata

of dyskeratosis, also may also develop marrow

failure. Genetic variants usually are monoallelic

(one mutated allele and one wild-type allele),

and TERT, TERC, and RTEL1 are the genes

usually affected. Telomere loss in these cases

is often less intense than in classic dyskeratosis

congenita. As a result of inadequate telomerase

function, the stem cell pool is limited in size

and in its ability to regenerate. There is insufficient production of erythrocytes, platelets,

and granulocytes with anemia, thrombocytopenia, and leukopenia of peripheral blood

and low marrow cellularity (Fig. 469-5). The

most typical presentation is moderate aplastic

anemia after a long history of macrocytic mild

to moderate anemia and/or thrombocytopenia,

with preservation of leukocyte numbers. A

comprehensive personal and family history is

important, querying especially for blood count

abnormalities and cytopenia as well as lung

and liver disease; early hair graying, while not

specific to telomeropathies, strongly suggests

telomere disease in the appropriate context.

Immunosuppressive therapy is generally

ineffective in these patients, and they may be more susceptible to

pulmonary or hepatic complications after hematopoietic stem cell

transplant.

Myeloid Neoplasms Some patients diagnosed with myelodysplastic syndrome (Chap. 102) or acute myeloid leukemia (Chap. 104) have

a family history of bone marrow failure or of other myeloid neoplasms.

One of the genetic causes for myeloid neoplasia predisposition is a

telomere defect, and these disorders are now classified together by the

World Health Organization as “myeloid neoplasms associated with

telomere biology disorders.” Telomere length measurement may be

confounded by the presence of circulating immature cells, which may have very

short telomeres, precluding appropriate

test interpretation.

Pulmonary Fibrosis Pulmonary

fibrosis appears in ~20% of children

with dyskeratosis congenita, and conversely, ~10–15% of patients with

idiopathic pulmonary fibrosis (Chap.

293) or familial pulmonary fibrosis have an etiologic telomerase gene

mutation. Most pulmonary fibrosis

patients, regardless of mutation status,

have short telomeres for their age but

not as short as in dyskeratosis congenita. How telomere erosion causes

pulmonary fibrosis is unclear, but it

might prevent adequate proliferation

and regeneration of pneumocytes type

II. Idiopathic pulmonary fibrosis due

to a telomere disease usually manifests

after the fourth decade of life, with a

restrictive pattern on pulmonary function testing associated with decreased

diffusion capacity for carbon monoxide (DLCO) and a diffuse “honeycomb”

appearance on high-resolution CT

Telomere Disease

3683CHAPTER 469

FIGURE 469-3 Clinical consequences of telomere diseases. Telomere dysfunction affects a variety of organs:

cerebellum, eyes, lungs, liver, skin, gastrointestinal tract, and the bone marrow.

(Fig. 469-5). Histopathology of biopsied lung most commonly

shows usual interstitial pneumonia. The pulmonary clinical presentation in telomere disease is indistinguishable from idiopathic

pulmonary fibrosis, except that those with an underlying telomere

defect may have cryptic hepatic cirrhosis, macrocytosis, cytopenias,

and a family history of lung, liver, or bone marrow disease. Pulmonary arteriovenous malformation leading to right-to-left shunting

is observed in patients with pulmonary fibrosis due to telomere

disease. Patients with idiopathic pulmonary fibrosis or familial pulmonary fibrosis should have leukocyte telomere length assayed and,

if telomeres are short, undergo screening for mutations in telomereassociated genes and telomeres; however, telomere length may be

normal in some cases despite the presence of pathogenic mutations.

TERT, TERC, RTEL1, and PARN are the most commonly mutated

genes.

Liver Disease Genetic telomere defects

may cause hepatic cirrhosis (Chap. 344),

nodular regenerative hyperplasia of the liver,

nonalcoholic fatty liver disease (Chap. 343),

and hepatocellular carcinoma (Chap. 82).

Hepatocytes of most patients with cirrhosis

have very short telomeres. Eroded telomeres

limit hepatocyte proliferation, especially

upon chronic injury. Additionally, hepatocytes with short telomeres display abnormal

metabolic pattern and defective mitochondrial function. Abnormal liver pathology

may be uncovered incidentally during the

evaluation of telomeropathy patients with

aplastic anemia or pulmonary fibrosis, but

cirrhosis also may be the sole or most prominent clinical presentation of a telomere

defect. A minority of individuals with cirrhosis associated with virus B or C infection

or alcohol-associated liver disease may carry

a telomere-associated gene mutation. Liver

histopathology is variable, but cirrhosis is

usually associated with inflammation and

inflammatory cells (Fig. 469-5), increased

iron deposit, positivity for CD34 in sinusoid

endothelial cells, and widening of hepatocyte

plates. Defective telomere maintenance may

increase susceptibility of the liver to environmental challenges, such as alcohol and viruses, increasing the risk for

developing severe hepatic disease in mutation carriers.

■ TELOMERE LENGTH MEASUREMENT

Length of telomeres can be accurately measured in peripheral blood

leukocytes by commercial laboratories. Of several methods available,

flow–fluorescent in situ hybridization (FISH) and quantitative realtime polymerase chain reaction (qPCR) are most widely utilized. Both

methods have advantages and limitations and require high-quality

samples, usually fresh or freshly processed, as cell death and DNA degradation impact the accuracy of testing. Results are usually expressed

as leukocyte telomere length in kilobases. However, the interpretation

of length must account for patient age due to physiologic telomere loss.

A normal range for telomeres is available for each year of age, with

length longest at birth and shortening at 40–60 base pairs per year

(Fig. 469-1). For each age bracket, the percentile curves are calculated,

and a given patient’s test result is interpreted in the context of normal

age variation: telomeres below the tenth percentile for age are defined

as “short,” and telomeres below the first percentile are considered “very

short” (Fig. 469-4).

Telomeres may also be short in groups of patients with some chronic

conditions, such as cardiovascular disease or diabetes. However, in

these settings, telomere erosion is not assumed to be etiologic, but

rather a consequence of chronic inflammation; telomere testing is

not known to have clinical utility and is not recommended. Likewise,

telomere length tests have no known clinical utility in the assessment

of aging and longevity or as a basis for therapeutic interventions absent

a diagnosis of telomere disease.

Flow-FISH uses a fluorescent-labeled nucleotide probe specific for

telomere repeats in order to estimate telomere content in an individual

cell by flow cytometry. It has the advantage of determining telomere

length in individual cells and in leukocyte subpopulations (neutrophils,

lymphocytes, monocytes); lymphocyte telomere shortening is more

specific for telomere diseases than in other cells. However, flow-FISH

requires intact cells for analysis, which are not always available, and

neutrophils are susceptible to damage during processing, freezing, and

thawing.

qPCR utilizes telomere-binding modified primers to measure

telomere content in comparison to a housekeeping gene in the whole

leukocyte population and thus does not require intact cells. qPCR

Age, years

Telomere length, kb

0

0

2

4

6

8

10

12

14

16

10 20 30 40 50 60 70 80 90

1%

10%

50%

90%

99%

TINF2

DKC1

TERT

TERC

RTEL1

Other genes

Peripheral blood leukocyte’s telomere

length in telomere diseases

FIGURE 469-4 Telomere length measurement in dyskeratosis congenita. Telomeres

shorten with aging, and solid curves represent the percentiles for age in healthy

subjects. Telomeres are considered “short” when below the tenth percentile and

very short when below the first percentile. In patients with dyskeratosis congenita,

telomeres are usually below the first percentile, regardless of the gene lesion.

3684 PART 16 Genes, the Environment, and Disease

FIGURE 469-5 Pathologic manifestations of telomere diseases. A. In the bone marrow, telomere erosion predisposes to aplastic anemia, characterized by an empty

hematopoietic marrow replaced by fat (hematoxylin and eosin). B. In the liver, telomere attrition predisposes to cirrhosis (hematoxylin and eosin). C. Telomere shortening

may also result in nodular regenerative hyperplasia of the liver (reticulin stain). D. In the lungs, telomere dysfunction predisposes to pulmonary fibrosis mainly in the

subpleural regions, which may be detected by high-resolution CT scan.

provides an estimate of the average telomere length of a given sample

without determining telomere length in individual cells. Good DNA

quality is essential for adequate qPCR testing and automation or semiautomation required for clinical purposes, as variability in conditions

among batches may result in interassay variation.

■ GENETIC TESTING

When a patient with a suspected telomeropathy has short or very

short telomeres, genetic screening for mutations in genes involved in

telomere maintenance and biology is indicated (Table 469-1).

Genetic testing should be restricted to patients with suspected telomere

disorders. Sanger sequencing has been used for screening purposes but

has been replaced by next-generation sequencing, and for telomerase

complex and shelterin genes, commercial panels are available. Mutations may be biallelic or triallelic, involving two genes (especially in

dyskeratosis congenita), but in most cases of aplastic anemia, myelodysplastic syndrome, acute myeloid leukemia, idiopathic pulmonary

fibrosis, and hepatic cirrhosis, only one allele is mutated. Thus, it is

crucial to appropriately interpret genetic screening results, as several

rare singleton polymorphisms of unknown significance have been

identified in large cohorts of healthy individuals. In silico analysis,

mutation location, and functional studies are utilized before declaring

a mutation pathogenic.

Genetic counseling is necessary after screening, as the inheritance

pattern may be autosomal dominant, mutation penetrance is highly

variable, and phenotypes may be diverse even within a pedigree. Potential family stem cell donors must be screened before transplantation to

ensure that they do not have mutations.

TREATMENT

Telomere Disease

Patients with severe aplastic anemia due to telomere disease may

undergo allogeneic hematopoietic stem cell transplant when a

suitable donor is available. Treatment-related mortality may be

increased due to pulmonary and hepatic complications, for which

reduced-intensity conditioning regimens may be advantageous.

Lung transplant for pulmonary fibrosis is feasible but often not performed due to coexisting cytopenias, especially thrombocytopenia,

and other comorbidities. Additionally, patients with pulmonary

fibrosis associated with telomere disease have a poorer outcome

after lung transplant and with nontransplant therapies. Similarly,

there is no specific treatment for the liver in telomere disease;

liver transplant has been attempted in rare cases. Telomeropathy

patients should be advised to avoid toxins (metal dust, busulfan,

amiodarone), ionizing radiation, cigarette smoke, and alcohol as

possibly harmful.

Long-term therapy with androgens may mitigate telomere attrition and even elongate leukocyte telomere length in humans. In

research trials, danazol improved blood counts in marrow failure

patients and reduced transfusion requirement.

■ FURTHER READING

Blackburn EH et al: Human telomere biology: A contributory and

interactive factor in aging, disease risks, and protection. Science

350:1193, 2015.

Calado RT, Young NS: Telomere diseases. N Engl J Med 361:2353,

2009.

Collins J, Dokal I: Inherited bone marrow failure syndromes. Hematology 20:433, 2015.

Gruber HJ et al: Telomeres and age-relatd diseases. Biomedicines

9:1335, 2021.

Gutierrez-Rodrigues F et al: Pathogenic TERT promoter variants in

telomere diseases. Genet Med 21:1594, 2019.

Holohan B et al: Telomeropathies: An emerging spectrum disorder. J

Cell Biol 205:289, 2014.

Newton CA et al: Telomere-related lung fibrosis is diagnostically

heterogeneous but uniformly progressive. Eur Respir J 48:1710, 2016.

Savage SA, Bertuch AA: The genetics and clinical manifestations of

telomere biology disorders. Genet Med 12:753, 2010.

Swaminathan AC et al: Lung transplant outcomes in patients with

pulmonary fibrosis with telomere-related gene variants. Chest

156:477, 2019.

Townsley DM et al: Bone marrow failure and the telomeropathies.

Blood 124:2775, 2014.

Townsley DM et al: Danazol treatment for telomere diseases. N Engl

J Med 374:1922, 2016.

Gene- and Cell-Based Therapy in Clinical Medicine

3685CHAPTER 470

Gene therapy is a novel area of therapeutics in which the active agent

is a nucleic acid sequence rather than a protein or small molecule. One

of the most powerful concepts in modern molecular medicine, gene

therapy has the potential to address a host of diseases for which there

are currently no available treatments. Because delivery of naked DNA

or RNA to a cell is an inefficient process, most gene therapy is carried

out using a vector, or gene delivery vehicle. These vehicles have generally been engineered from viruses by deleting some or all of the viral

genome and replacing it with the therapeutic gene of interest under the

control of a suitable promoter, but nonviral delivery vehicles such as

lipid nanoparticles have also been used (Table 470-1). Gene therapy

strategies can thus be described in terms of three essential elements:

(1) a gene delivery vehicle; (2) a gene to be delivered, sometimes called

the transgene; and (3) a physiologically relevant target cell to which the

DNA or RNA is delivered. The series of steps in which the vector and

donated DNA or RNA enter the target cell and express the transgene

is referred to as transduction. Gene delivery can take place in vivo, in

which the vector is directly injected into the patient, or, in the case

of hematopoietic, immune, and some other target cells, ex vivo, with

removal of the target cells from the patient, followed by return of the

gene-modified autologous cells to the patient after manipulation in

the laboratory. The latter approach effectively combines gene transfer

techniques with cellular therapies (Chap. 484).

Currently approved gene therapies rely on transfer of DNA, but

investigational RNA-based therapies have gained ground rapidly, partly

as a key component of CRISPR-based gene editing (guide RNAs) and,

more recently, as the active agent in two SARS-CoV-2 vaccines. This

chapter will focus primarily on approved therapies (Table 470-2), with

some discussion of investigational therapies in late-phase development

and earlier trials of historical interest.

Clinical trials of gene therapy have been under way since 1990;

the first gene therapy product to be licensed in the United States or

Europe was approved in 2012 (see below). Given that vector-mediated

gene therapy is arguably one of the most complex therapeutics yet

developed, typically consisting of both a nucleic acid and a protein

470 Gene- and Cell-Based

Therapy in Clinical

Medicine

Katherine A. High, Marcela V. Maus

component, this time course from first clinical trial to licensed product

is noteworthy for being similar to those seen with other novel classes

of therapeutics, i.e., monoclonal antibodies. Thousands of subjects

have been enrolled in gene transfer studies or have received approved

products, and serious adverse events have been rare. Some of the initial

trials were characterized by an overabundance of optimism and a failure to be appropriately critical of preclinical studies in animals; in addition, it was in some contexts not fully appreciated that animal studies

are only a partial guide to safety profiles of products in humans (e.g.,

insertional mutagenesis and human immune responses to the vector).

Clinical experience and laboratory research led to a more nuanced

understanding of the risks and benefits of these new therapies and

more sophisticated selection of disease targets. Currently, gene therapies are being developed for a variety of disease entities (Table 470-3).

GENE TRANSFER FOR GENETIC DISEASE

Gene transfer strategies for genetic disease generally involve gene

addition therapy, an approach characterized by transfer of the missing

gene to a physiologically relevant target cell. However, other strategies

are possible, including supplying a truncated form of the gene with

comparable biologic activity (e.g., a gene encoding B domain-deleted

factor VIII for hemophilia A); supplying a gene that achieves a similar biologic effect through an alternative pathway (e.g., utrophin in

place of dystrophin for Duchenne’s muscular dystrophy); supplying an

antisense oligonucleotide to alter splicing of a transcript to generate

a functional protein (e.g., nusinersen in the treatment of spinal muscular atrophy); or downregulating a harmful effect through a small

interfering or short hairpin RNA. From a therapeutic standpoint,

gene therapies for genetic disease fall into two distinct categories: first,

they may provide treatment for diseases that have hitherto lacked any

pharmacologic therapies; or second, they may provide an alternative to

complex medical regimens that are frequently characterized by significant nonadherence due to the burden of treatment (e.g., monthly red

blood cell transfusions and iron chelation in transfusion-dependent β

thalassemia).

Gene therapy for genetic disease requires long-term expression of

the transgene. Two distinct strategies are available to achieve this goal:

one is to transduce stem cells with an integrating vector, so that all

progeny cells will carry the donated gene; and the other is to transduce

long-lived, postmitotic cells, such as skeletal muscle or neurons. In

the case of long-lived cells, integration into the target cell genome is

unnecessary. Instead, because the cells are nondividing, the donated

DNA, even if stabilized predominantly in an episomal form, will give

rise to expression for the life of the cell. This latter approach mitigates

risks related to integration and insertional mutagenesis.

TABLE 470-1 Characteristics of Commonly Used Gene Delivery Vehicles

VIRAL NONVIRAL

FEATURES RETROVIRAL LENTIVIRAL ADENOVIRAL AAV LIPID NANOPARTICLES

Viral genome RNA RNA DNA DNA N/A

Cell division requirement Yes G1

 phase No No No

Packaging limitation 8 kb 8 kb 8–30 kb 5 kb 10 kb or more

Immune responses to vector Few Few Extensive Few Few

Genome integration Yes Yes Poor Poor May be used to package

either RNA or DNA

Long-term expression Yes Yes No Yes Transient for RNA

Main advantages Persistent gene transfer

in dividing cells

Persistent gene transfer in

transduced tissues; no history

of insertional oncogenesis;

can add desired promoter

Highly effective in

transducing various

tissues

Elicits few

inflammatory

responses,

nonpathogenic

Limited immune

responses against the

vector

Main disadvantages History of insertional

oncogenesis (occurred

in multiple cases); only

used ex vivo; gene

silencing

Might induce oncogenesis in

some cases (not yet observed);

only used ex vivo

Viral capsid elicits strong

immune responses

Limited packaging

capacity

Currently available

reagents predominantly

target the liver

Abbreviations: AAV, adeno-associated virus; N/A, not applicable.