Добавил:
Sekretar
kiopkiopkiop18@yandex.ru
t.me/Prokururor I Вовсе не секретарь, но почту проверяю
Опубликованный материал нарушает ваши авторские права? Сообщите нам.
Вуз:
Предмет:
Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_104_библиотеки_им_акад_М_И_Перельмана
.pdf
The molecular basis ofbone marrow failure syndromes andred cell enzymopathies 153
PIGT*
Cell membrane
https://t.me/med1917
fibroblasts partial, GPI deficiency. The precise molecular
mechanism of this disease has been delineated. C > G substitution disrupts binding of the generic transcription factor Sp1
to a GC- rich box in the core promoter of PIGM. Unlike in red
cells, where PIGM transcription is independent of Sp1 binding to its core promoter, in B cells, PIGM transcription is
dependent upon Sp1 binding, hence the divergent GPI
expression across cell lineages. Impaired Sp1 binding leads to
reduced histone acetylation in the promoter of PIGM and in
turn transcriptional repression by nucleosomal compaction,
recruitment of the Polycomb repressor complex and establishment of a bivalent chromatin state. As such, PIGMassociated IGD represents the first Mendelian disease whose
pathogenesis is attributed to Polycomb- mediated bivalent
chromatin domain transcriptional repression. Importantly,
histone hypoacetylation is reversible. Specifically, in the presence of histone deacetylase inhibitors such as sodium
butyrate, histone acetylation, PIGM transcriptional activity,
GPI biosynthesis, and surface GPI- linked protein expression
can all be restored invivo as well as invitro. This remarkable
effect was highlighted by the complete abrogation of complex,
life- long, and treatment- refractory epilepsy in a child with
inherited GPI deficiency treated with sodium butyrate.
In a small number of patients with variable clinical phenotypes, whole exome sequencing analysis has identified germline mutations in several other genes of the GPI biosynthetic
pathway (Plate 11.2). As with PIGM, these patients have
homozygous or bi- allelic mutations that result in partial deficiency of the corresponding protein. Clinical manifestations
comprise epilepsy, learning difficulties, and multiple organ
abnormalities, but not BMF, with the exception of one
reported case of PNH caused by a germ- line mutation in one
PIGT allele accompanied by a somatic mutation in the second allele in neutrophils.
Inherited bone marrow failure
syndromes
Fanconi anemia
The incidence of FA is estimated at 1:360,000in the general
population. Early studies indicated that FA was a genetically
heterogeneous disease, a notion confirmed by the identification, through the use of somatic cell hybridization and
latterly genome sequencing, of 21 complementation groups/
genes (Table 11.2). The overall frequency of heterozygotes
for FA mutant genes in the general population is estimated at
1in 300. In Ashkenazi Jews and in the Afrikaans population
of the South Africa, it is much higher (1in 100 and 1in 89,
respectively), most likely as a result of founder effects.
Clinical aspects The most common clinical manifestation is
gradual onset of BMF. BMF typically appears by the age of
10years (median age 7 years; range, birth to 31 years) and is
often heralded by isolated thrombocytopenia, macrocytosis,
EtNP
PIGO
M
EtNP
EtNP
Inositol
Plate 11.2 The structure of GPI–protein (protein–EtNP-6Manα12Manα1-6Manα1-4GlcNα1-6myoinositol-phospholipid) is shown.
GPIbiosynthesis takes place mostly in the endoplasmic reticulum.
Thefirst step involves the addition of acetylglucosamine (GlcN) to
phosphatidylinositol (inositol-P). Synthesis of the GPI anchor proceeds
with the serial addition of a glycan moiety consisting of three mannose
(M) molecules each modified by phosphoethanolamine (EtNP). Through
a transpeptidation reaction, proteins with the appropriate carboxyterminal amino acid motif are attached covalently to the terminal EtNP.
The GPI–protein complex subsequently travels to the cell surface, where
the protein becomes anchored to the lipid bilayer through GPI. In PNH,
PIG-A, a protein encoded by the X-linked gene PIGA and a member of
a multi-subunit enzymatic complex that catalyzes the first step (i.e.
addition of GlcN to inositol-P), is somatically mutated in one or few
HSCs. As a result, very little GPI is synthesized, or none at all, with
consequent severe deficiency of GPI-anchored proteins on the surface
of the mutated HSCs and their progeny. InInherited GPI deficiency,
germline hypomorphic mutations in PIGL,PIGM, PIGN, PIGV, PIGO,
PGAP2, or PGAP3 result in variable GPI deficiency. Germline mutations
in PIGA are embryonically lethal in men, with the exception of two
families. Here, mono- allelic PIGA mutations resulted in a truncated PIGA
protein with sufficient residual function to allow survival of the affected
individuals, but causing severe congenital abnormalities and intellectual
disability. For PIGT, a case of a germline mutation in one allele with a
tissue- specific somatic mutation in neutrophils has been reported.
Abbreviations: P- Phosphatidyl; M- mannose; GlcN- glucosamine;
EtNP- phosphoethanolamine.
M
M
GlcN
P
protein
PGAP2
PGAP3
PIGVPIGN
PIGM
PIGL
PIGA
GPI
maturation in
Golgi
and increased hemoglobin F levels (the latter indicative of
stress erythropoiesis). Congenital abnormalities are present
in two- thirds of patients and comprise skeletal abnormalities
(most commonly of the radius and thumb), skin lesions
(hyperpigmentation, café- au- lait spots), renal and urinary
tract malformations, and gonadal dysfunction. However, the
本书版权归John Wiley & Sons Inc.所有
154 Molecular Hematology
DN
damage
(ICLS)
https://t.me/med1917
Table 11.2 Identity ofthe Fanconi anemia (FA) genes andproteins
Approximate
FA genes Inheritance
FANCA AR 65
FANCB X linked recessive 2
FANCC AR 12
FANCD1/BRCA AR 2
FANCD2 AR 4
FANCE AR 1
FANCF AR 2
FANCG AR 8
FANCI AR 1
FANCJ/BRIP1 AR 2
FANCL AR <1
FANCM AR <1
FANCN/PALB2 AR <1
FANCO/RAD51C
FANCP/SLX4 AR <1
FANCQ/ERCC4/XPF AR <1
FANCR/RAD51 AD <1
FANCS/BRCA1
FANCT/UBE2T
FANCU/XRCC2
FANCV/REV7/MAD2L2 AR <1
FANCW AR <!
a
Loss of function mutations in these genes have been
associatedwith Fanconi- like congenital abnormalities, but
notwith BMF.
a
AR <1
a
a
a
AR <1
AR <1
AR <1
frequency of FA (%)
clinical spectrum is even wider, as it includes congenital
defects of the gastrointestinal system, heart, and central
nervous system. Specific syndromes encompassing
Fanconianemia are VACTERL- H (Vertebral, Anal, Cardiac,
Trachea- esophageal fistula, Esophageal/duodenal atresia,
Renal, Limb, Hydrocephalus) association and PHENOS
(Pigmentation, small- Head, small- Eyes, Neurologic,
Otologic, Short stature).
Patients with FA have an unusually high risk of developing
treatment- resistant MDS and AML, estimated at 52% by the
age of 40 years. Furthermore, the risk of a variety of solid
tumors, especially squamous cell carcinomas of the skin,
head, neck, and anogenital region, is several times higher
than in the general population. Patients with mutations in
BRCA2 or PALB2 usually develop AML or embryonal tumors
in the first few years of life. While FA results from bi- allelic
mutations, heterozygous mutations in BRCA1, BRCA2,
PALB2, and RAD51C are associated with increased risk of
solid tumors, in particular breast and ovarian cancer. This
has implications for first- degree relatives of an individual
with FA caused by one of these specific complementation
groups.
Cellular phenotype and function of the FA proteins FA proteins
constitute the “FA pathway,” a DNA damage- response pathway responsible for the repair of inter- and intra- strand DNA
cross- links (ICL), either occurring spontaneously or induced
by agents like mitomycin C or diepoxybutane (Plate11.3). ICL
prevent the separation of the two DNA strands, leading to
F
FC complex
C
B
E
G
M
A
T
L
+Ub
ID complex
A
ATR
+P
Plate 11.3 Molecular pathogenesis of Fanconi anemia. The FA pathway of DNA repair is activated in response to intra- or inter- strand DNA
cross- links. First, the DNA sensor ATR phosphorylates the Fanconi core complex (FCC) and ID complexes. Subsequently, FCC monoubiquitinates
FANCD2 and FANCI. The ID complex is recruited to the site of DNA damage and acts in concert with DNA repair proteins. Specifically, FANCP/SLX4
and FANCU/ERCC4 catalyze unlooping of the interstrand crosslinks (ICL) allowing translesional synthesis by Polζ, one component of which is
FANCV/REV7. The final phase of ICL repair is mediated by the remaining DNA repair proteins including FANCD1/BRCA2, FANCN/PALB2, FANCJ/
BRIP2, and FANCS. BRCA1 along with FANCR/RAD51 and its paralogs: FANCO/RAD51c and FANCU/XRCC2. Ultimately, the coordinated functions
of these proteins lead to the repair of the DNA cross- link through nucleotide excision repair, translesional synthesis, and homologous
recombination.
Ub
Ub
I
D2
P
P
Downstream effectors of DNA repair
Polζ
P
V
Ub
I
P
Q
Ub
D2
P
N
J
S
Ub
Ub
I
D2
P
D1
P
R O
U
本书版权归John Wiley & Sons Inc.所有
The molecular basis ofbone marrow failure syndromes andred cell enzymopathies 155
https://t.me/med1917
replication fork arrest and cell cycle arrest in late S phase.
Completion of the cell cycle is not possible unless ICL are
repaired. FA proteins, organized in several groups functioning
in a linear fashion, are essential for thesuccessful resolution of
the stalled DNA replication at thesite of the ILC. The FA core
complex (FCC), comprising nineproteins (FANCA, B, C, E, F,
G, M, and T), receives signals of DNA damage through phosphorylation by ATR, a kinase sensor of DNA damage. This
promotes binding of the FCC to the damaged DNA through
FANCM and FAAP24. FANCL, through its ubiquitin ligase
activity, monoubiquitinates FANCD2 and FANCI, the two
proteins that make up a structure termed the ID complex.
FANCL interacts with UBE2T, a ubiquitin- conjugating
enzyme that is required for monoubiquitination of FANCD2.
Ubiquitination of the ID proteins along with their phosphorylation by ATR are required for their specific localization to
the site of DNA damage, where they interact with a further
family of proteins. The finding that these proteins, corresponding to FANCD1, FANCN, and FANCJ complementation groups, were in fact identical to BRCA2 and its associated
PALB2 and BRIP1 proteins, respectively, was an exciting discovery linking the FA pathway to an already established process of DNA repair. In concert with a number of other proteins
of the DNA repair machinery (e.g. RAD51, RAD51C, XRCC2),
BRCA1 and BRCA2 are involved in different repair processes
that include nucleotide excision repair, trans- lesional synthesis, and homologous recombination, all of which are required
for removal and repair of ICL and restarting of DNA replication. Nucleotide excision repair is also performed by two proteins recently found to be mutated in DKC: SLX4 and ERCC4.
These form a complex that facilitates nucleolytic incisions at
the ICL by the endonuclease ERCC4, paving the way for
homologous recombination. Taken together, a mutation
affecting any one of the FANC proteins impairs DNA repair
resulting in genomic instability in hematopoietic stem cells
and in turn, bone marrow failure.
Molecular genetics FA is an autosomal recessive disorder with
the exception of FANCB, which is X-
linked and FANCR,
which is autosomal dominant. The most characteristic cellular feature of FA cells is the formation of DNA doublestrand breaks on exposure to DNA inter- and intra- strand
adducting agents (clastogens) such as mitomycin C and diepoxybutane. The invitro response to clastogens has made it
possible to test cells from different patients for their ability to
cross- correct each other’s defect by somatic cell fusion (hence
the 21 complementation groups).
Mutations of FANCA account for about 60% of FA cases
and are spread throughout the gene. None of the mutant
alleles is common and few have been encountered more than
once. Mutations in the FANCC gene account for about
10–15% of FA cases. The IVS4 + A → T and del322G muta-
tions comprise more than 75% of FANCC mutations. The
IVS4 + A → T allele is found in Ashkenazi Jews at a
polymorphic frequency (1in 80), and it is responsible for
85% of theFA cases in this population. Patients with IVS4 or
exon 14mutations tend to have earlier onset of hematological complications (BMF and MDS/AML), and to have a
shorter survival compared with patients with exon 1mutations or patients with FANCA or FANCG-
related FA.
Mutations in the FANCG gene are found in about 10% of FA
cases. The stop codon mutation E105X accounts for 44% of
mutations in German FANCG patients. It is interesting that
although bi- allelic null mutations are very frequent in FANC
genes of FCC, only hypomorphic mutations have been identified in FANCD2 (3–6% of all cases of FA), highlighting a
more important role of the ID complex that is not restricted
to ICL correction only but also encompasses the repair of
ionizing irradiation- induced double- strand DNA damage.
Consistent with this, FANCD2 patients, despite the hypomorphic nature of the mutations, appear to develop the most
severe form ofFA, with multiple developmental abnormalities and early development of BMF and malignancy. The
prevalence of mutations in other FANC genes is very low
(<2%).
FA and cancer An important role of the FA pathway in the
biology of sporadic cancers is increasingly being recognized.
Epigenetic silencing of FANC genes has been identified in
various solid tumors and in acute leukemias. In some cases,
this correlates with their sensitivity to chemotherapeutic
agents causing DNA cross- links such as cisplatin, cyclophosphamide, and melphalan. In a small proportion of patients
with AML, as well as those with head and neck squamous
cell and gynecological cancers, somatic mutations in FANCA
have been identified, but their functional significance is
unknown. Therefore, studying the functional status of the
FA pathway in different tumors may offer valuable leads to
therapeutic choices.
Diagnosis The clastogen test (outlined above) remains a useful clinical diagnostic test; however, screening for pathogenic
mutations in the FANC genes has now entered routine clinical practice. With this approach, genetic counseling, prenatal
and pre-
implantation diagnosis are now feasible for most
families with affected children. In areas with a large
Ashkenazi Jewish population (e.g. New York City), it is
realistic to carry out screening for polymorphic FANC alleles
on a wider population basis, so that it can be offered to all
couples at risk.
Treatment The conventional management of FA focuses
on the consequences of BMF and includes hematopoietic
growth factors, blood product support, and androgens.
About half of the patients respond to androgens initially but
often suffer from significant side- ef fects, including androgeninduced hepatic adenomas. Eventually, all patients become
refractory.
本书版权归John Wiley & Sons Inc.所有
156 Molecular Hematology
T
d
https://t.me/med1917
HSCT from an HLA- identical unaffected sibling or from
alternative sources is currently the only therapeutic approach
that can successfully achieve long- term correction of BMF
and possible prevention of MDS and AML. Best results, with
reduced short- and long- term treatment- related mortality,
are obtained with fludarabine- based, reduced- intensity,
non- myeloablative conditioning HSCT regimens that do
notinclude irradiation (to reduce the risk of solid tumors).
T- cell depletion of the graft is employed to minimize
graft- versus- host disease. However, even after HSCT,
patients remain at increased risk of developing solid tumors
and thus require close long- term follow- up as well as preventative strategies such as Human Papilloma Virus (HPV)
vaccination.
Identification of the FA genes has paved the way for gene
therapy for patients with FA. There is experimental and
clinical evidence that HSCs with corrected phenotype have a
survival and growth advantage over uncorrected cells and
can support long- term hematopoiesis. Successful transfer of
FA genes to a small number of autologous HSCs should
therefore be adequate to ameliorate the severity of BMF.
Dyskeratosis congenita and the telomere
biology disorders (TBD)
DKC is another rare (incidence approximately 1in 1 million), genetically heterogeneous, inherited disorder with
BMF as a major feature and a 10% risk of malignancy. DKC
is primarily a disorder of telomeres, the ends of chromosomes. Telomeres are made of several megabases of DNA
consisting of identical nucleotide repeats (TTAGGG), and
of a protein complex essential for chromosome integrity
(Plate 11.4). The main clinical features of DKC are lacy
reticulated skin, nail dystrophy, and mucosal leukoplakia in
the first years of life and later, the development of BMF.
Other clinical manifestations include developmental delay,
short stature, ocular, dental, and skeletal abnormalities,
hyperhidrosis, hyperkeratinization of the palms and soles,
bullae on minimal trauma, hair loss, nail loss, sometimes
gonadal failure, and features of premature aging. Patients
with DKC are also at risk of pulmonary fibrosis and hepatic
failure. The clinical spectrum of DKC is variable among
patients and dynamic with age. Some patients manifest only
minimal physical features and normal hematopoiesis, while
others have the classical triad of skin, nail, and mucosal
abnormalities coupled with early- onset BMF. Like FA, DKC
predisposes to malignancy, in particular MDS/AML and
squamous cell carcinomas of the head, neck, and anogenital
tract. The median age of onset is 35 years for MDS and
37years for solid cancers.
Molecular genetics and pathogenesis Several genes have been
identified to cause DKC when mutated (Table 11.1).
Mutations occur de novo, and the diseases they cause are
inherited as X- linked recessive, autosomal dominant, or
autosomal recessive traits.
The X- linked form of DKC was the first to be identified
and characterized. It results mostly from missense mutations
in the housekeeping gene DKC1. Mutations in DKC1, which
maps to Xq28 (like G6PD and hemophilia A), account for
about 40% of all DKC cases. The Hoyerall–Hreidarsson
syndrome, a rare disorder characterized by severe growth
Telomerase complex
TYMS
ZCCHC8
TERT
GGGATT
CCCUAA
WRAP53/
TCAB1
(telomere trafcking)
3'
(nucleotide synthesis)
Plate 11.4 Schematic representation of human proteins involved in telomere maintenance. TERC encodes a non- coding RNA and no
protein. This RNA template interacts with TERT and other members of the telomerase complex to elongate telomeres by adding hexanucleotide
tandem repeats to the ends of chromosomes. The allied roles of the components of the CST and shelterin complexes are shown. Shaded proteins are
those that, when affected by germ- line mutations, cause DKC. PAPD5 oligoadenylates immature TERC and thus targets it for exosome degradation.
本书版权归John Wiley & Sons Inc.所有
PARN
NAF1
GAR
NOP10
NHP2
DKC1
PAPD5
(destabilizes TERC)
5'
TERC
(RNA template)
RTEL1 RPA1
Telomere replication
and stability
processing
ACD/
TPP1
POT1
TERC
3'
TIN2/TINF2
CTC1
(C strand synthesis)
Shelterin complex
(protection of telomere ends)
T
T
R
R
F
F
1
2
STN1
TEN1
CST complex
RAP1
SNM1B/Apollo
stran
G strand
Telomeric
DNA
DNA
C strand
NPM1
MDM4 USB1
(p53 regulator) (RNA modiers)
The molecular basis ofbone marrow failure syndromes andred cell enzymopathies 157
https://t.me/med1917
failure, severe immunodeficiency, cerebellar abnormalities,
and BMF, is in most cases allelic to DKC1. Three unrelated
cases of Hoyerall–Hreidarsson syndrome caused by biallelic
mutations in Apollo/SNM1B, have also been reported.
Apollo- deficient patient cells exhibit chromosome instability
but normal telomere lengths.
Dyskerin, the protein product of DKC1, is homologous
tothe yeast protein Cbf5p. Cbf5p, in conjunction with the H/
ACA class of small nucleolar RNAs, is required for the pseudouridylation (i.e. conversion of uridine to pseudouridine)
of pre- ribosomal RNA (rRNA), a modification essential for
ribosome biogenesis. Although defective pseudouridylation
and ribosome biogenesis have been observed in a hypomorphic DKC1mouse model, other studies failed to demonstrate
any qualitative or quantitative defect of rRNA in DKC
patients. Instead, it was shown that dyskerin binds, via its
domain called PUA, to an H/ACA domain of the telomerase
RNA component (TERC), that is the template used by its
protein component, the telomerase reverse transcriptase
(TERT). Thus, dyskerin is intimately linked to telomerase in
its crucial function of telomere maintenance during cell division. Most DKC1 mutations map to the PUA domain, linking
DKC to abnormal RNA binding.
Telomerase, as just outlined above, is a ribonucleoprotein
complex, and dyskerin is one of the several proteins that by
interacting directly or indirectly with TERC, preserve its
stability and its ability to function as a template for TERT.
Telomerase activity is highest in rapidly dividing cells (e.g. in
hematopoietic progenitors, in tumor cells, during organogenesis and in early life). The critical evidence that DKC is a
disease of telomere dysfunction was provided by the finding
that heterozygous mutations in the TERT and TERC genes
themselves cause a disease virtually indistinguishable from
that caused by DKC1 mutations. Once again, not unlike FA,
the multitude of genes whose mutations can give rise to a
dyskeratosis phenotype relates to the complexity of the
telomere maintenance/servicing machinery (see Table 11.1
and Plate 11.4). NOP10 and NHP2, members of the H/ACA
snoRNPs family, localize like DKC1 itself to nucleoli and to
Cajal bodies in the nucleus. WRAP53 assists with the transportation of TERT to the Cajal bodies, and PARN facilitates
the interaction between TERC and the telomere. RTEL1
encodes a DNA helicase required to unwind the T loop
structure, a prerequisite for telomere lengthening, whereas
CTC1 forms part of the CST complex that caps and protects
telomeres. Finally, TINF2 and ACD encode proteins that
form part of the so-
called shelterin complex, which binds to
telomeric DNA and provides stability. Mutations in TINF2
have been associated with the Revesz syndrome, a form
of DKC characterized by bilateral exudative retinopathy.
Patients with mono- allelic mutations are affected, and therefore the condition is designated as autosomal dominant;
however, those with bi- allelic mutation have far more severe
disease. Thus, all types of DKC are related to a common
molecular pathogenetic pathway, namely the maintenance
of telomere length. In keeping with this notion, all types of
blood cells in DKC patients have significantly shorter telomeres than age-
matched controls. Shortening of telomeres to a
critical length can result in either (i) growth arrest and apoptosis – hence the development of BMF; or (ii) neoplastic
transformation– hence the increased propensity to epithelial
tumors. The identification of TYMS deficiency, characterized by short telomeres and the first case of DKC caused
by digenic mutations, shows that telomere maintenance
depends on nucleotide metabolism. Ribosome stress also
occurs downstream of telomere maintenance thus the clinical phenotype results from the interplay of several pathophysiological processes.
The study of the genetics of DKC has revealed at least three
additional interesting features. First, in families with TERC,
TERT, and TINF2 mutations, DKC has tended to show earlier
onset and more severe manifestations in successive generations: a phenomenon known as genetic anticipation. In keeping with this, the rate of loss of telomere length was higher in
successive generations within the same DKC family (just as
had been observed in Te r c −/− mice). Second, heterozygous
germ- line mutations of TERT or TERC have been detected in
a small minority (<5%) of patients who had been initially
diagnosed with AA. In many cases, these mutations were not
obviously deleterious for the function of telomerase invitro;
in some cases, they were not detected in the parents of index
cases and in others they did not segregate with the disease in
the respective families. A possible interpretation of these
findings is that mild TERC and TERT mutations may predispose to BMF. However, this develops only upon exposure to a
trigger, such as an immunological attack. Third, in patients
with pathogenic mutations of TERT, TERC, and DKC1,
somatic mosaicism has been demonstrated, whereby the
mutation is found in DNA from skin fibroblasts but not in
DNA from peripheral blood cells. The presumed mechanism
is genetic reversion through back- mutation in a single hematopoietic cell. The self- corrected cell would have a significant
selective advantage, in some cases sufficient to repopulate the
bone marrow, an example of nature’s gene therapy. Indeed,
missense heterozygous mutations in RPA1 cause a TBD
encompassing bone marrow failure, myelodysplastic syndrome, T-
and B- cell lymphopenia, pulmonary fibrosis, or
skin manifestations. Somatic genetic rescue has been identified in hematopoietic cells due to anacquired truncating cis
RPA1 mutation or due to uniparental isodisomy of 17p with
loss of the mutant allele, and acquisition of these genetic
events coincided with stabilized blood counts.
Adult- onset telomere biology disorders are increasingly
being recognized and 25% of patients manifest over the
ageof 40 years. These may present with BMF, pulmonary
fibrosis, or liver failure and are associated with heterozygous
variants in specific genes: TERT, TERC, and RTEL1. 1–4% of
AA cases and 2.7% of MDS harbor mutations in TERC or
TERT. Above the age of 40 years, patients are often oligosymptomatic and lack the classical DKC triad.
本书版权归John Wiley & Sons Inc.所有
158 Molecular Hematology
https://t.me/med1917
Most patients with DKC die before the age of 30 years,
predominantly from complications of BMF, opportunistic
infections, pulmonary complications, and malignancy.
The prognosis with TINF2 mutations is especially poor,
while most patients with AD mutations on TERC or TERT
are alive at 50 years.
Diagnosis DKC is characterized by very short telomeres,
defined as <1% of age- matched normal controls, in the
majority of leukocyte subsets. Telomere shortening is the
primary defect rather than a reflection of loss of stem cell
reserve. Measurements are performed by multi- parameter
flow cytometry fluorescence in situ hybridization (flowFISH). In lymphocytes, sensitivity and specificity for DKC
are 97% and 91%, respectively. Genetic testing is desirable to
confirm diagnosis, but pathogenic germline mutations are
detected in only about 70% of patients with a clinical diagnosis of DKC, either because the panel of genes is incomplete or
because some causative genes are still unknown.
Telomere lengths can also be measured by terminal restriction fragment analysis by Southern blotting or quantitative
PCR- based assays (Single- Telomere Length Analysis:
STELA); however, these are not widely used due to the ease,
accuracy, and reproducibility of flow- FISH.
Treatment As for FA, there is no definitive treatment forDKC
and allogeneic HSCT is the only curative option forsevere
BMF or MDS/leukemia. HSCT in DKC has an increased risk
of graft failure, graft- versus- host disease, pulmonary fibrosis, hepatic cirrhosis, and veno- occlusive disease.
Conditioning requires a reduced intensity, alkylator- free
approach to mitigate the risk of secondary cancers. If a
related donor or matched unrelated donor is unavailable, one
can try therapy with androgens, namely 100 mg danazol on
alternate days. The mechanism of action of these agents is
thought to be direct up- regulation of TERT gene expression,
by binding to the estrogen response element in its promotor,
with consequent increased telomerase activity and improved
telomere length. Although androgens induce a trilineage
response in blood counts in 70% of patients, these responses
are lost with time in the majority of patients. With the exception of adolescent boys, androgens are contra-
indicated
in children due to masculinization and risk of hepatic
adenomas.
In contrast to AA and FA respectively, TPO agonists have
no effect in DC and lentiviral gene therapy is precluded by
the fact that telomerase overexpression leads to cancer. Gene
correction by means of CRISPR/Cas9- mediated gene editing
may lead to the future development of gene therapy for DKC
as well as other heritable bone marrow failure syndromes.
PAPD5 is a non- canonical polymerase that oligoadenylates
and destabilizes TERC. PAPD5 inhibitors stabilize TERC
and restore telomere length in iPSCs from patients with
dyskeratosis congenita, thus holding promise as a new therapy for DKC and TBD.
Diamond–Blackfan anemia
Diamond–Blackfan anemia (DBA) is a rare (1–2 in
100,000live births) inherited syndrome with isolated anemia
and erythroblastopenia as the main features, although rarely
with disease progression, patients may develop bi- or trilineage hypoplasia. Clinically DBA manifests itself in the
first months of life with macrocytic anemia, reticulocytopenia, increased hemoglobin F and elevated erythrocyte adenosine deaminase (eADA) levels. It is not known why the
anemia associated with DBA rarely manifests during fetal
life. The bone marrow is normocellular with characteristic
paucity of erythroblasts (<5% of nucleated cells). Growth
retardation is seen in about 30% of patients, and other developmental abnormalities (including skeletal, cardiac, and
urogenital defects) in 50% of patients. There is also an
increased risk (∼20% by age 46 years) of MDS/AML or solid
tumors, mainly osteogenic sarcoma.
Molecular genetics and pathogenesis In most cases, DBA is
caused by a mono- allelic, loss- of- function ribosomal protein
(RP) gene mutations. DBA is therefore one of the “ribosomopathies,” a group of inherited BMF syndromes also comprising Shwachman–Diamond syndrome, X- linked DKC,
and cartilage hair hypoplasia (Table 11.1). In 55–60% of
cases, mutations are de novo, whereas in the remainder, they
are transmitted in an autosomal dominant fashion. The first
pathogenic genetic variant identified was a heterozygous
mutation in RPS19, the most common (25% of cases) gene
involved in DBA. The finding that the whole RPS19 gene is
deleted in some cases of DBA, strongly argued that haploinsufficiency, rather than a dominant negative effect, is the
underlying pathogenetic mechanism in DBA. As well as
being a structural component of the mature small ribosomal
subunit (40S; large subunit is 60S), RPS19 is also required in
the nucleolus for the correct processing and cleavage from
the precursor rRNA of the 18S fragment, which is the main
rRNA constituent of 40S. In the same process, which is
RPS19-
independent, precursor rRNA is also cleaved to 5.8S
and 28S rRNA which, along with the independently transcribed 5S rRNA, are the constituents of the large ribosomal
subunit. Haploinsufficiency of RPS19 and of the other RP
genes linked to DBA results in defective formation of the
ribosomal subunits and eventually defective formation of the
mature ribosome. How impaired ribosome function might
lead to the relatively selective phenotype of erythroid hypoplasia is not clear. It is possible that this is related to the
extraordinary demand that hemoglobin production imposes
on the translational apparatus. Specifically, there is evidence
that inadequate globin synthesis leads to excess heme
本书版权归John Wiley & Sons Inc.所有
The molecular basis ofbone marrow failure syndromes andred cell enzymopathies 159
https://t.me/med1917
production in erythroid progenitors and in turn to ferroptosis. Selective impaired translation of the mega- erythroid
transcription factor GATA1has also been demonstrated in
animal and cellular models of DBA, though GATA1levels
are relatively preserved in patients with DBA caused by large
ribosomal subunit mutations suggesting genotype- specific
mechanisms of anemia. Many ribosomal proteins, including
RPS19, have been found to participate in extra- ribosomal
functions. For example RPS19 might affect cell cycle progression through its interaction with the serine/threonine
kinase Pim- 1, whereas RPL11 and RPL5 stabilize and activate p53 through an inhibitory physical association with the
p53 regulator HDM2.
Since the identification of RPS19, several other RP genes
have been implicated in DBA11.4(Table 11.3). A wide variety
of mutations have been described including missense, nonsense, splicing, and whole allele deletions. Of note, DBA
resulting from an RP gene deletion can occur in the context of
other syndromes, for example the microdeletion 3q29 syndrome, in which the deleted region encompasses RPL35A.
Mutations in the promoter region of RPS19 have also been
Table 11.3 Genes mutated inDiamond–Blackfan anemia
Gene name % of cases of DBA
RPS19 25
RPL5 6.6
RPS26 6.4
RPL11 4.8
RPL35/35A 3
RPS10 2.6
RPS24 2
RPS17 1
RPS7 <1
RPS29 <1
RPL26 <1
RPL15 <1
RPL31 <1
RPS27 <1
RPL4 <1
RPL8 <1
RPL9 <1
RPL17 <1
RPL18 <1
RPL26 <1
RPS28 <1
RPS15/15A <1
RPS20 <1
RPL27 <1
GATA-
1 <1
TSR2 <1
HEATR3 <1
observed, and it is likely that more variants in regulatory areas
will be implicated in DBA, as knowledge of the function of
the non- coding genome expands. In the last few years, two
X- linked genes associated with DBA- like disease have been
discovered. GATA- 1 and TSR2 (an RP chaperone) and bial-
lelic variants have been described in HEATR3, which is neces-
sary for RPL5nuclear import. Improved characterization of
the genetic lesions underlying the development of DBA has
led to the identification of genotype- phenotype correlations
including a later age of presentation with anemia and higher
hemoglobin but higher preponderance of birth defects in
DBA caused by RPL5 or RPL11 variants. Despite this pro-
gress, we are yet to fully elucidate the molecular basis of the
clinical heterogeneity observed in DBA, including the
remarkable variable penetrance within families.
Diagnosis Diagnostic criteria for DBA include the presence
of typical hematological and clinical features and identification of a causative gene mutation. The presence of congenital
anomalies, age < 1 year, raised eADA and macrocytosis all
point toward a diagnosis of DBA, rather than transient erythroblastopenia of childhood, the main differential. This is an
important distinction as the latter condition usually resolves
spontaneously within a few months.
In specialized DBA centers, genetic diagnosis using
targeted next- generation sequencing of RP genes has
replaced traditional Sanger sequencing. This approach,
complemented by additional techniques such as multiplex
ligation- dependent probe amplification for validation of
computationally identified deletions, as well as the identification of new candidate RP genes, have led to an improvement in the pick- up rate of mutations from 40% to around
70% of patients (Table11.3).
Interestingly, a recent study discovered that 8 of 173
patients with presumed DBA but no pathogenic RP gene
mutation, harbored in fact mitochondrial DNA deletions
similar to those seen in the Pearson marrow- pancreas syndrome. Therefore, while we have addressed each of the
inherited bone marrow failure syndromes separately, it is
important to recognize that they share a triad of clinical features: anemia, physical abnormalities, and predisposition to
cancer. Given the overlap between these conditions and the
accessibility of genetic testing in routine clinical practice, a
child presenting with congenital anemia should be ideally
tested for multiple conditions, using a panel encompassing
multiple genes. Furthermore, cases in which a mutation is
not identified by targeted multigene assays, are candidates
for whole genome sequencing with the aim of novel gene discovery, as exemplified by the United Kingdom’s Genomics
England 100,000 Genomes Project.
Treatment Blood transfusion, combined with iron chelation,
is the mainstay of treatment for moderate to severe anemia
本书版权归John Wiley & Sons Inc.所有
160 Molecular Hematology
https://t.me/med1917
inDBA. After regular immunizations with live vaccines are
completed during infancy, most patients are started on highdose glucocorticoids (GC). Initially, 80% of patients respond,
with a surge in reticulocytes followed by a rise in hemoglobin, to the extent that the patient may become transfusionindependent. However, not infrequently GC treatment is
discontinued, either because the response is lost (this can
happen with puberty or pregnancy), or because at the dose
required to maintain therapeutic benefit, the side effects are
unacceptable. In the long term, only 20–40% of patients
remain on a very low dose of GC; a further 10–20% seem to
go into spontaneous remission by the age of 25 years. Patients
who are steroid- resistant, or those with severe iron overload
or progressive bone marrow failure must be considered for
allogeneic HSCT. Ideally, this should be performed before
the age of 10 years. At present, there is convincing evidence
to recommend both sibling and matched unrelated donors,
with donor stem cells derived from bone marrow, sometimes
in combination with umbilical cord blood.
Recent experimental evidence indicated that, through
activation of the rapamycin- sensitive mTOR/RPS6 pathway,
the amino acid L- leucine enhances ribosomal translational
activity in DBA cells with RPS19 mutations. As yet, a clinically effective treatment for patients with DBA has failed to
evolve from these findings. One of the most clinically important unsolved riddles in DBA is the mechanism of action of
GC and how to translate this to new therapies that directly
target erythropoiesis in steroid- resistant patients.
As supportive therapy improves and patients with DBA
can expect longer lives, it becomes increasingly important to
understand the drivers of malignant transformation in a subset of patients. The relevance of ribosome biology to diseases
far more common than DBA has been highlighted recently
by the discovery of RP gene mutations in hematological
malignancies (specifically in T- cell acute lymphoblastic
leukemia and in chronic lymphocytic leukemia). In many
cases, these somatically acquired mutations interfere with
the function of the tumor suppressor p53.
Red cell enzyme deficiencies
Inherited abnormalities of red cell enzymes, red cell enzymopathies, are a distinct set of genetic disorders with one impor-
tant clinical manifestation in common, namely chronic
hemolytic anemia. This section deals with those enzymopathies affecting red cell metabolism for which the molecular
basis has been elucidated (Table 11.4). We do not discuss
conditions in which an enzyme abnormality is also expressed
in red cells but the main clinical manifestations are elsewhere
(e.g. the porphyrias, galactosemia, Lesch–Nyhan syndrome).
All of these defects are rare to very rare. For the sake of
brevity, we will give a brief overview of enzymopathies and
then focus on deficiencies of pyruvate kinase (PK), glucose6- phosphate dehydrogenase (G6PD), and pyrimidine
5′- nucleotidase – representative of disorders of glycolysis,
the pentose phosphate pathway and nucleotide metabolism,
respectively.
Clinical features: In these disorders, hemolytic anemia occurs
with varying degrees of severity. It is not unusual for the
presentation to be in the guise of severe neonatal jaundice
that may require exchange transfusion; if the anemia is
less severe, it may present later in life, or it may even
remain asymptomatic and be detected incidentally when a
blood count is performed for unrelated reasons. The
spleen is often enlarged. When other systemic manifestations occur, they involve the central nervous system,
sometimes entailing severe mental retardation, or the neuromuscular system, or both.
Diagnosis: The diagnosis of hemolytic anemia is usually not
difficult, thanks to the triad of normo- macrocytic anemia,
reticulocytosis, and hyperbilirubinemia. Enzymopathies
should be considered in the differential diagnosis of any
chronic Coombs- negative hemolytic anemia. A definitive
diagnosis can be made only by demonstrating the deficiency of an individual enzyme by a quantitative assay. For
the sake of economy, it is sensible to carry out these rather
laborious tests in order of frequency of occurrence of the
various enzymopathies: first glucose- 6 phosphate dehydrogenase (G6PD), then pyruvate kinase (PK), then
glucose 6- phosphate isomerase, and so on (Table 11.4).
If a particular molecular abnormality is already known in
the family, then of course, one could test directly for that at
the DNA level, bypassing the need for enzyme assays.
Asfor other multigenic heritable disorders, the advent of
high throughput sequencing technologies allows simultaneous analysis of multiple genes implicated in glycolytic
enzyme deficiencies, thus simplifying the genetic diagnosis of these disorders. Nevertheless, enzymatic assays
should still be performed to validate candidate pathogenic
mutations.
Molecular pathophysiology: Most of the glycolytic enzymes
involved are housekeeping enzymes present, by definition,
in all cells. Therefore one might expect that a severe reduction in activity of any of these might have generalized
clinical manifestations. However, we can identify at least
two reasons why red cells are more severely affected.
Firstly, red cells have a much more limited metabolic
machinery than most other somatic cells; if a particular
enzyme is deficient, other cells may cope by the use of
alternative or surrogate metabolic pathways. Secondly,
mature red cells lack nuclei and organelles and thus are
not capable of protein synthesis. Therefore if a particular
本书版权归John Wiley & Sons Inc.所有
The molecular basis ofbone marrow failure syndromes andred cell enzymopathies 161
https://t.me/med1917
Table 11.4 Synopsis of red cell enzymopathies
Isoenzymeb
characteristic of
Enzyme (abbreviation)
red cells
a
Prevalence of
enzyme
deficiency
Main clinical features
associated with
enzyme deficiency
c
Benefit from
splenectomy
Chromosomal
d
localization
Adenylate kinase (AK) 1 Very rare HA, CNS Partial 9q34.1
Aldolase A Very rare HA, myopathy 16q22–q24
Cytochrome b
2, 3- Diphosphoglycerate mutase
reductase Rare Pseudocyanosis, CNS 22q13.31–qter
5
Very rare Polycythemia 7q31–q34
(DPGM)
e
Enolase
Glucose 6- phosphate
1 (α) Very rare HA 1pter–p36.13
B Common HA None Xq28
dehydrogenase (G6PD)
Glucose 6- phosphate isomerase
Rare HA, NM, CNS Partial 19q13.1
(GPI)
γ- Glutamylcysteine synthetase
f
(GLCLC)
Glutathione peroxidase (GSH-
Px) Very rare
Very rare HA, CNS 6p12
g
g
?
3q11–q12
Glutathione reductase (GSR) Very rare HA 8p21
Glutathione synthetase (GSS) Very rare HA, CNS 20q11.2
Glyceraldehyde 3- phosphate
dehydrogenase (GAPD)
e
Very rare HA 12p13.31–p13.1
Hexokinase (HK) R (I) Very rare HA Partial 10q22
Lactate Dehydrogenase- A (LDHA) Very rare Myopathy 11p15
Monophosphoglycerate mutase
(PGAM- B)
Phosphofructokinase (PFK)
h
B Very rare Myopathy and
10q25.3
myoglobinuria
M Very rare HA, myopathy 12q13
L 21q22.3
Phosphoglycerate kinase (PGK) 1 Very rare HA, CNS, NM Partial Xq13
Pyrimidine 5′- nucleotidase (P5′N1) Rare HA Partial 7p15–p14
Pyruvate kinase (PK) R
i
Rare HA Partial 1q21
Triosephosphate isomerase (TPI) Very rare HA, CNS, NM None 12p13
a
We have listed all enzymes in the intermediary metabolism of red cells for which, to the best of our knowledge, the corresponding cDNA/
gene has been cloned.
b
No entry in this column means that there are no known isoenzymes; therefore it is assumed that the same enzyme type is present in all
tissues.
c
CNS, central nervous system involvement; HA, hemolytic anemia; NM, neuromuscular manifestations.
d
Data available only on some patients.
e
Since no mutations have yet been reported, there is no formal proof that HA associated with this enzyme deficiency is due to mutation of
the corresponding gene.
f
γ- Glutamylcysteine synthetase consist of two subunits: a catalytic subunit and a regulatory subunit. The data concerning the catalytic subunit
are shown here.
g
Enzyme reported to be low in some nutritional deficiencies, but clear evidence that inherited deficiency of glutathione peroxidase exists is
lacking.
h
PFK in normal red cells consists of a mixture of the five tetrameric species that can be formed from random association of the M (muscle)
and L (liver) highly homologous subunits (i.e. M4, M3L, M2L2, ML3, L4).
i
The red cell form of PK called R is produced by the gene encoding the L (liver) subunit. Because a different promoter is used, the size of liver
PK is 543 amino acids.
Source: Modified from Luzzatto L, Notaro R. (1998) Red cell enzymopathies. In: Jameson JL (ed.). Principles of Molecular Medicine. Clifton,
NJ:Humana Press, pp.197–207, with permission.
本书版权归John Wiley & Sons Inc.所有
162 Molecular Hematology
https://t.me/med1917
enzyme is made highly unstable by a mutation, other cell
lineages can compensate by increased enzyme synthesis,
but red cells cannot.
Management: There is no specific treatment for these condi-
tions. Patients with moderate anemia may require occasional blood transfusion when they experience
exacerbations of the anemia due to increased rate of
hemolysis or to decreased red cell production secondary
to infection (the most extreme example being aplastic crisis from parvovirus infection). Patients with chronic
severe anemia may require regular blood transfusion therapy and in some patients, splenectomy has been beneficial
(Table11.4).
Pyruvate kinase deficiency
Pyruvate kinase (PK) deficiency is the most common defect
of the glycolytic pathway causing chronic non- spherocytic
hemolytic anemia (CNSHA). It has an estimated frequency
of 1 in 20,000 in people of European ancestry and a
higher frequency in the Old Order Amish population of
Pennsylvania. The clinical phenotype is variable, from
hydrops fetalis (non- immune) to mild anemia presenting in
the second or third decade. Therefore under- diagnosis of PK
deficiency occurs at both ends of the clinical spectrum.
Pathophysiology. PK catalyzes the second ATP- producing
step of the glycolytic pathway; thus, it accounts for nearly 50%
of the red blood cell’s chemical energy in the form of ATP (see
Figure11.1 for an overview of glycolysis). The activity of PK,
the last reaction in the anaerobic glycolytic pathway, is allosterically regulated by the product of one of the first reactions
of the pathway, namely fructose- 1,6- bisphosphate (FBP).
There are 4 PK isoforms (M1, M2, L, and R) encoded by two
separate genes (PK- M and PK- LR). Alternative splicing of the
PK- LR transcript encodes PKL and PKR in the liver and red
blood cells, respectively. In PK deficiency, like in other glycolytic enzymopathies, a shortage of energy supply leads to
shortened red blood cell lifespan and red blood cell destruction mainly in the spleen. Glycolytic enzymes are normally
present in cells in considerable excess, and therefore the 50%
loss of enzyme activity seen in heterozygotes does not become
limiting for red cell survival; thus, heterozygotes for PK deficiency do not have hemolytic anemia. Indeed, the disease
shows an autosomal recessive pattern of inheritance. Affected
patients are either homozygotes or compound heterozygotes
with bi-
allelic mutations. From the hematologic point of view,
it is interesting that PK- deficient patients, after splenectomy,
have reticulocytosis to a degree (up to 50%) very seldom seen
in any other human condition. This has led to the suggestion
that there may be a specific mechanism for the spleen to
remove PK- deficient reticulocytes from the circulation.
Another interesting feature of PK deficiency is that
bisphosphoglycerate (BPG) levels are high (up to three
2,3times normal). This produces a right shift in the hemoglobin O2 dissociation curve, thus increasing O2 delivery to tissues,
which partially compensates for anemia.
Molecular genetics. Over 200mutations have been implicated
in PK deficiency and, as is the case for other glycolytic
enzymes, most mutations are of the missense type, causing
single amino acid replacements. This is important because
the low level of residual enzyme activity can still support
some metabolic flow through the glycolytic pathway, which
explains how red cells survive in the circulation, even though
their lifespan is reduced. With respect to how precisely individual missense mutations reduce enzyme activity, we must
consider at the protein level several possible mechanisms:
(i)In the majority of cases, loss of activity is probably due to
decreased stability of the protein. In such cases, we would
predict that, compared to erythrocytes, other cells, for example hepatocytes, might be much less affected, because they
can compensate for decreased stability through increased
synthesis of the enzyme. (ii) In some cases, the amino acid
replacement, being within or near the active center of the
enzyme, may affect substrate binding (Km), or catalytic rate
(K
), or both. (iii) In some cases, the allosteric regulation by
cat
FBP may be affected (altered Ka). In cases (ii) and (iii) not
only red cells, but other cells, in which the rate of glycolysis is
critical, will be affected as well. Among severe missense mutations of this type, one of the most common in Caucasians is
994A (Gly332Ser), which can, when homozygous, cause
intrauterine death. Cloning of the PKLR gene in an expression vector and purification of recombinant normal and
mutant human PKR enzyme has facilitated studying the biochemical impact of single amino acid substitutions. Of course,
as with most inherited disorders, the correlation between
genotype and phenotype based on the molecular properties
of the enzyme, is only a first approximation, because other
inherited and acquired factors regularly come into play,
whereas most PK mutations are sporadic, it was found in subSaharan Africa that one polymorphic mutation (E277K) has
heterozygote frequencies up to 7%, and it has been suggested
it may have been biologically selected by malaria.
Diagnosis. PK deficiency is suggested by the constellation of
clinical features, and hematological indices; unlike in other
glycolytic enzymopathies, red cell morphology can be also
suggestive, with a multitude of cells displaying spiculae on
their surface. As with all autosomal recessive disorders, there
may be no family history. Diagnosis is confirmed by finding
in red cells reduced PK enzymatic activity, which is usually
10–25% of normal in patients with bi- allelic mutations. Of
note, since routine enzyme tests measure V
without regard
max
to Km or Ka, enzymatic activity correlates poorly with the
severity of hemolysis, and may be even normal. Mutation
本书版权归John Wiley & Sons Inc.所有
Соседние файлы в папке Библиотека им академика М.И. Перельмана