Medical Genetics
05 疾病的单基因遗传
Monogenic Inheritance
Medical Genetics
single-gene disorder or
monogenic disorder
Some disorders result when a
mutation causes the product of a single
gene to be altered or missing,These
disorders are inherited in simple patterns
similar to or identical with those described
by Mendel for certain discrete
characteristics in garden peas,Therefore,
it’s also called Mendelian diseases,
Medical Genetics
Basic Pattern of Single Gene
Inheritance
Autosomal Dominant
Autosomal Recessive
X-linked Dominant
X-linked Recessive
Y-linked
Medical Genetics
1,Pedigree and Proband
Humans are unique among
organisms in many ways,but one
way which is near and dear to a
geneticist's heart is that humans are
not susceptible to genetic
experimentation,
Medical Genetics
The study of inherited Mendelian
traits in humans must rely on
observations made while working
with individual families,
Medical Genetics
Classical cross fertilization
breeding experiments as performed
by Mendel are not allowed in humans!
Human geneticists are not allowed to
selectively breed for the traits they
wish to study!
Medical Genetics
One of most powerful tools in
human genetic studies is pedigree
analysis,
Medical Genetics
pedigree
They are graphic representations
of a family tree which show the
biological relationship of the index
case,or proband or propositus to the
rest of the individuals,
A family tree diagram that shows
how a particular genetic trait or
disease has been inherited,
Medical Genetics
When human geneticists first
began to publish family studies,they
used a variety of symbols and
conventions,Now there are agreed
upon standards for the construction
of pedigrees,
Medical Genetics
Symbols
Medical Genetics
Medical Genetics
Medical Genetics
2,autosomal dominant inheritance
The pattern of autosomal
dominant inheritance is perhaps the
easiest type of Mendelian inheritance
to recognize in a pedigree,One dose
of the mutant gene,one mutant
allele,is all that is required for the
expression of the phenotype,
Medical Genetics
There are three reasons why an
individual with an autosomal
dominant disease should always be
considered as being a heterozygote
until proven otherwise,
Medical Genetics
A,The disease is usually rare,with only
about 1/10,000 individuals affected as an
order of magnitude,Affected individuals
are most likely to come from affected by
normal matings,The normal parent is
homozygous recessive,thus assuring that
each product of the mating has at least
one normal gene,
Medical Genetics
B,In the extremely rare instances where
two affected individuals have mated,the
homozygous affected individuals usually
are so severely affected they are not
compatible with life,The exceptions are
the autosomal dominant diseases caused
by the somatic expansion of trinucleotide
repeat sequences (e.g.,Huntington's
disease) that we will study later,
Medical Genetics
C,The mating of very closely related
individuals,the most likely way for
two affected individuals to know each
other,is forbidden in our society,
Medical Genetics
With the understanding that
almost all affected individuals are
heterozygotes,and that in most
matings involving a person with an
autosomal dominant trait the other
partner will be homozygous normal,
there are four hallmarks of
autosomal dominant inheritance,
Medical Genetics
There are four hallmarks of autosomal
dominant inheritance,
(1) Except for new mutations,which are rare in nature and
extremely rare on examination pedigrees,and the
complexities of incomplete penetrance to be discussed later,
every affected individual has an affected biological
parent,There is no skipping of generations,
(2) Males and females have an equally likely chance of
inheriting the mutant allele and being affected,The
recurrence risk of each child of an affected parent is
1/2,
Medical Genetics
(3) Normal siblings of affected individuals
do not transmit the trait to their offspring,
(4)The defective product of the gene is
usually a structural protein,not an enzyme,
Structural proteins are usually defective when
one of the allelic products is nonfunctional;
enzymes usually require both allelic products to
be nonfunctional to produce a mutant phenotype,
Medical Genetics
THE PUNNET SQUARE
In 1910,Punnett developed a
simple method of depicting the
possible genotypes one could get
from various matings,We call it the
Punnett Square,
Medical Genetics
Suppose a father is heterozygous
for an autosomal dominant gene,
with allele D,the mutant dominant
allele,and allele d,the recessive
normal allele,He can produce two
types of gametes,D and d,Suppose
also his wife is homozygous normal,
having both d alleles,The Punnett
Square is constructed as follows,
Medical Genetics
One gamete comes from each parent to
produce the genotype of the offspring,Two
out of the four possible combinations are
affected; two out of four are normal,
Medical Genetics
Sample Pedigree
Medical Genetics
neurofibroma
Medical Genetics
marfan‘s syndrome
Medical Genetics
3,AUTOSOMAL RECESSIVE INHERITANCE
The first,and most important,
thing to remember about autosomal
recessive inheritance is that most,if
not all,affected individuals have
parents with normal phenotypes,
Medical Genetics
Suppose the disease affects
one in ten thousand live births,a
good order of magnitude estimate for
most autosomal recessive diseases,
That would make the heterozygote
frequency in the population one in
fifty (see population genetics for
calculations),
Medical Genetics
The likelihood of two affected persons mating
would be 1/10,000 x 1/10,000 or 1/100,000,000,
By chance alone there might be two such matings
in the Unites States,but no more than 2,The
likelihood of an affected and a heterozygote
mating would be 1/10,000 x 1/50 x 2(since either
parent could be the affected) or 1/250,000,The
likelihood of two heterozygotes (heterozygotes
are usually called "carriers") mating is 1/50 x
1/50 or 1/2500,more than 99% of all possible
matings,
Medical Genetics
The Punnett Square for autosomal
recessive diseases with an affected child
in the family almost always looks like the
following,
Medical Genetics
Where the father and mother
are both Dd (dd is the recessive
affected individual,Dd the
heterozygous carrier individual,and
DD the homozygous normal
individual),The Punnet Square
shows the origin of the famous
Mendelian ration of 3/4 normal to
1/4 affected,
Medical Genetics
For most autosomal recessive
diseases,but not all,the heterozygote
cannot be distinguished from the normal
homozygote,In the normal phenotype
categories of offspring in the above
Punnett Square (Dd and DD produce the
same normal phenotype),please note that
two of the three are heterozygotes
(carriers); one of the three is homozygous
normal,
Medical Genetics
Within the normal siblings of an
affected individual the probability of
being a carrier is 2/3,
Medical Genetics
There are five hallmarks of autosomal
recessive inheritance,
(1) Males and females are equally likely to be affected,
(2) On average,the recurrence risk to the unborn sibling
of an affected individual is 1/4,
(3) The trait is characteristically found in siblings,not
parents of affected or the offspring of affected,
(4) Parents of affected children may be related,The
rarer the trait in the general population,the more
likely a consanguineous mating is involved,
(5) The trait may appear as an isolated (sporadic) event
in small sibships,
Medical Genetics
Sample pedigree
Medical Genetics
When consanguinity is involved,
i.e.,matings between related
individuals,in the production of an
affected child the assignment of
probabilities changes,especially in
the rarer autosomal recessive
diseases,
Medical Genetics
? Sample pedigree
Medical Genetics
amaurotic idilcy
Medical Genetics
Wilson’s disease
Medical Genetics
4,X-LINKED DOMINANT INHERITANCE
When an X-linked gene is said to
express dominant inheritance,it
means that a single dose of the
mutant allele will affect the
phenotype of the female,A recessive
X-linked gene requires two doses of
the mutant allele to affect the female
phenotype,
Medical Genetics
Affected father x normal mother,Affected mother x normal father,
Medical Genetics
The following are the hallmarks of X-
linked dominant inheritance,
(1)The trait is never passed from father to son,
(2)All daughters of an affected male and a normal
female are affected,All sons of an affected male and
a normal female are normal,
(3)Matings of affected females and normal males
produce 1/2 the sons affected and 1/2 the daughters
affected,
(4)Males are usually more severely affected than
females,The trait may be lethal in males,
(5)In the general population,females are more likely to
be affected than males,even if the disease is not
lethal in males,
Medical Genetics
Sample pedigree
Medical Genetics
incontinentia pigmenti
Medical Genetics
vitamin D-resistant Rickets
Medical Genetics
5,X-LINKED RECESSIVE INHERITANCE
Everyone has heard of some X-linked
recessive disease even though they are,in
general,rare,Hemophilia,Duchenne
muscular dystrophy,Becker muscular
dystrophy,and Lesch-Nyhan syndrome
are relatively rare in most populations,but
because of advances in molecular genetics
they receive attention in the media,
Medical Genetics
More common traits,such as
glucose-6-phosphate dehydrogenase
deficience or color blindness,may occur
frequently enough in some populations to
produce a few affected females,However,
their effect on individuals is rarely life
threatening and medical intervention is
not needed,Pedigree 7 shows one typical
inheritance pattern for a rare X-linked
recessive disease,
Medical Genetics
Medical Genetics
the hallmarks of X-linked recessive
inheritance
(1) As with any X-linked trait,the disease is never
passed from father to son,
(2) Males are much more likely to be affected than
females,If affected males cannot reproduce,only
males will be affected,
(3) All affected males in a family are related
through their mothers,
(4) Trait or disease is typically passed from an
affected grandfather,through his carrier
daughters,to half of his grandsons,
Medical Genetics
Sample pedigree
Medical Genetics
hemophilia
Medical Genetics
Medical Genetics
6,Y-linked
A gene on the Y chromosome,A
Y-linked gene is by necessity passed
from father to son,since the Y
chromosome can only be transmitted
by a man to his male progeny,
Medical Genetics
It has often been said that little is
known about whether specific genes
are or are not Y-linked,
Medical Genetics
a number of genes were known to be Y-linked
including,
ASMTY (acetylserotonin methyltransferase),TSPY
(testis-specific protein),IL3RAY (interleukin-3 receptor),
SRY (sex-determining region),TDF (testis determining
factor),ZFY (zinc finger protein),PRKY (protein kinase,Y-
linked),AMGL (amelogenin),CSF2RY (granulocyte-
macrophage colony-stimulating factor receptor,alpha
subunit on the Y chromosome),ANT3Y (adenine nucleotide
translocator-3 on the Y),AZF2 (azoospermia factor 2),
BPY2 (basic protein on the Y chromosome),AZF1
(azoospermia factor 1),DAZ (deleted in azoospermia),
RBM1 (RNA binding motif protein,Y chromosome,family 1,
member A1),RBM2 (RNA binding motif protein 2) and UTY
(ubiquitously transcribed TPR gene on Y chromosome),
Medical Genetics
Sample pedigree
Medical Genetics
Medical Genetics
7,SPECIAL FEATURES
Penetrance
The likelihood a given gene will
result in disease,For example,if half
(50%) of the people with the
neurofibromatosis (NF) gene have
the disease NF,the penetrance of
the NF gene is 0.5,
Medical Genetics
Expressivity
The consistency of a genetic
disease,For example,Marfan disease
shows variable expressivity,Some
persons with Marfan's merely have
long fingers and toes while others
have the full-blown disease with
dislocation of the lens and dissecting
aneurysm of the aorta,
Medical Genetics
Phenocopy
(1) An environmental condition that
imitates (copies) one produced by a
gene,
(2) The person who has an
environmentally-produced condition
that mimics one produced by a gene,
Medical Genetics
Genetic heterogeneity
Here a similar clinical picture may
be produced by different mutations
at the same locus or at different loci,
Retinitis pigmentosa may be caused
by both autosomal dominant or
recessive inheritance,
Medical Genetics
Anticipation
A remarkable phenomenon in which a genetic
disease appears earlier appearance and with
increased from with each succeeding generation,
Anticipation was once thought not to exist in
genetics,It was chalked off as a meaningless
statistical artifact,However,anticipation has now
been proven to occur in a large number of
important genetic disorders,including Huntington
disease and myotonic dystrophy,In molecular
terms,anticipation is due to the expansion of a
trinucleotide repeat sequence in the DNA,This
phenomenon also occurs in the fragile X
syndrome,the most common inherited form of
mental retardation,
Medical Genetics
Genomic imprinting
The phenomenon of parent-of-origin
gene expression,The expression of a
gene depends upon the parent who
passed on the gene,
Medical Genetics
For instance,two different disorders -
Prader-Willi syndrome and Angelman syndrome -
- are due to deletion of the same part of
chromosome 15,When the deletion involves the
chromosome 15 that came from the father,the
child has Prader-Willi syndrome,but when the
deletion involves the chromosome 15 that came
from the mother,the child has Angelman
syndrome,
Genomic imprinting plays a critical role in
fetal growth and development,Imprinting is
regulated by DNA methylation and chromatin
structure,
Medical Genetics
New mutations
Seen as isolated cases,
Increased paternal age may be
associated with new mutations,
Majority of Achondroplasics are
new mutations,
05 疾病的单基因遗传
Monogenic Inheritance
Medical Genetics
single-gene disorder or
monogenic disorder
Some disorders result when a
mutation causes the product of a single
gene to be altered or missing,These
disorders are inherited in simple patterns
similar to or identical with those described
by Mendel for certain discrete
characteristics in garden peas,Therefore,
it’s also called Mendelian diseases,
Medical Genetics
Basic Pattern of Single Gene
Inheritance
Autosomal Dominant
Autosomal Recessive
X-linked Dominant
X-linked Recessive
Y-linked
Medical Genetics
1,Pedigree and Proband
Humans are unique among
organisms in many ways,but one
way which is near and dear to a
geneticist's heart is that humans are
not susceptible to genetic
experimentation,
Medical Genetics
The study of inherited Mendelian
traits in humans must rely on
observations made while working
with individual families,
Medical Genetics
Classical cross fertilization
breeding experiments as performed
by Mendel are not allowed in humans!
Human geneticists are not allowed to
selectively breed for the traits they
wish to study!
Medical Genetics
One of most powerful tools in
human genetic studies is pedigree
analysis,
Medical Genetics
pedigree
They are graphic representations
of a family tree which show the
biological relationship of the index
case,or proband or propositus to the
rest of the individuals,
A family tree diagram that shows
how a particular genetic trait or
disease has been inherited,
Medical Genetics
When human geneticists first
began to publish family studies,they
used a variety of symbols and
conventions,Now there are agreed
upon standards for the construction
of pedigrees,
Medical Genetics
Symbols
Medical Genetics
Medical Genetics
Medical Genetics
2,autosomal dominant inheritance
The pattern of autosomal
dominant inheritance is perhaps the
easiest type of Mendelian inheritance
to recognize in a pedigree,One dose
of the mutant gene,one mutant
allele,is all that is required for the
expression of the phenotype,
Medical Genetics
There are three reasons why an
individual with an autosomal
dominant disease should always be
considered as being a heterozygote
until proven otherwise,
Medical Genetics
A,The disease is usually rare,with only
about 1/10,000 individuals affected as an
order of magnitude,Affected individuals
are most likely to come from affected by
normal matings,The normal parent is
homozygous recessive,thus assuring that
each product of the mating has at least
one normal gene,
Medical Genetics
B,In the extremely rare instances where
two affected individuals have mated,the
homozygous affected individuals usually
are so severely affected they are not
compatible with life,The exceptions are
the autosomal dominant diseases caused
by the somatic expansion of trinucleotide
repeat sequences (e.g.,Huntington's
disease) that we will study later,
Medical Genetics
C,The mating of very closely related
individuals,the most likely way for
two affected individuals to know each
other,is forbidden in our society,
Medical Genetics
With the understanding that
almost all affected individuals are
heterozygotes,and that in most
matings involving a person with an
autosomal dominant trait the other
partner will be homozygous normal,
there are four hallmarks of
autosomal dominant inheritance,
Medical Genetics
There are four hallmarks of autosomal
dominant inheritance,
(1) Except for new mutations,which are rare in nature and
extremely rare on examination pedigrees,and the
complexities of incomplete penetrance to be discussed later,
every affected individual has an affected biological
parent,There is no skipping of generations,
(2) Males and females have an equally likely chance of
inheriting the mutant allele and being affected,The
recurrence risk of each child of an affected parent is
1/2,
Medical Genetics
(3) Normal siblings of affected individuals
do not transmit the trait to their offspring,
(4)The defective product of the gene is
usually a structural protein,not an enzyme,
Structural proteins are usually defective when
one of the allelic products is nonfunctional;
enzymes usually require both allelic products to
be nonfunctional to produce a mutant phenotype,
Medical Genetics
THE PUNNET SQUARE
In 1910,Punnett developed a
simple method of depicting the
possible genotypes one could get
from various matings,We call it the
Punnett Square,
Medical Genetics
Suppose a father is heterozygous
for an autosomal dominant gene,
with allele D,the mutant dominant
allele,and allele d,the recessive
normal allele,He can produce two
types of gametes,D and d,Suppose
also his wife is homozygous normal,
having both d alleles,The Punnett
Square is constructed as follows,
Medical Genetics
One gamete comes from each parent to
produce the genotype of the offspring,Two
out of the four possible combinations are
affected; two out of four are normal,
Medical Genetics
Sample Pedigree
Medical Genetics
neurofibroma
Medical Genetics
marfan‘s syndrome
Medical Genetics
3,AUTOSOMAL RECESSIVE INHERITANCE
The first,and most important,
thing to remember about autosomal
recessive inheritance is that most,if
not all,affected individuals have
parents with normal phenotypes,
Medical Genetics
Suppose the disease affects
one in ten thousand live births,a
good order of magnitude estimate for
most autosomal recessive diseases,
That would make the heterozygote
frequency in the population one in
fifty (see population genetics for
calculations),
Medical Genetics
The likelihood of two affected persons mating
would be 1/10,000 x 1/10,000 or 1/100,000,000,
By chance alone there might be two such matings
in the Unites States,but no more than 2,The
likelihood of an affected and a heterozygote
mating would be 1/10,000 x 1/50 x 2(since either
parent could be the affected) or 1/250,000,The
likelihood of two heterozygotes (heterozygotes
are usually called "carriers") mating is 1/50 x
1/50 or 1/2500,more than 99% of all possible
matings,
Medical Genetics
The Punnett Square for autosomal
recessive diseases with an affected child
in the family almost always looks like the
following,
Medical Genetics
Where the father and mother
are both Dd (dd is the recessive
affected individual,Dd the
heterozygous carrier individual,and
DD the homozygous normal
individual),The Punnet Square
shows the origin of the famous
Mendelian ration of 3/4 normal to
1/4 affected,
Medical Genetics
For most autosomal recessive
diseases,but not all,the heterozygote
cannot be distinguished from the normal
homozygote,In the normal phenotype
categories of offspring in the above
Punnett Square (Dd and DD produce the
same normal phenotype),please note that
two of the three are heterozygotes
(carriers); one of the three is homozygous
normal,
Medical Genetics
Within the normal siblings of an
affected individual the probability of
being a carrier is 2/3,
Medical Genetics
There are five hallmarks of autosomal
recessive inheritance,
(1) Males and females are equally likely to be affected,
(2) On average,the recurrence risk to the unborn sibling
of an affected individual is 1/4,
(3) The trait is characteristically found in siblings,not
parents of affected or the offspring of affected,
(4) Parents of affected children may be related,The
rarer the trait in the general population,the more
likely a consanguineous mating is involved,
(5) The trait may appear as an isolated (sporadic) event
in small sibships,
Medical Genetics
Sample pedigree
Medical Genetics
When consanguinity is involved,
i.e.,matings between related
individuals,in the production of an
affected child the assignment of
probabilities changes,especially in
the rarer autosomal recessive
diseases,
Medical Genetics
? Sample pedigree
Medical Genetics
amaurotic idilcy
Medical Genetics
Wilson’s disease
Medical Genetics
4,X-LINKED DOMINANT INHERITANCE
When an X-linked gene is said to
express dominant inheritance,it
means that a single dose of the
mutant allele will affect the
phenotype of the female,A recessive
X-linked gene requires two doses of
the mutant allele to affect the female
phenotype,
Medical Genetics
Affected father x normal mother,Affected mother x normal father,
Medical Genetics
The following are the hallmarks of X-
linked dominant inheritance,
(1)The trait is never passed from father to son,
(2)All daughters of an affected male and a normal
female are affected,All sons of an affected male and
a normal female are normal,
(3)Matings of affected females and normal males
produce 1/2 the sons affected and 1/2 the daughters
affected,
(4)Males are usually more severely affected than
females,The trait may be lethal in males,
(5)In the general population,females are more likely to
be affected than males,even if the disease is not
lethal in males,
Medical Genetics
Sample pedigree
Medical Genetics
incontinentia pigmenti
Medical Genetics
vitamin D-resistant Rickets
Medical Genetics
5,X-LINKED RECESSIVE INHERITANCE
Everyone has heard of some X-linked
recessive disease even though they are,in
general,rare,Hemophilia,Duchenne
muscular dystrophy,Becker muscular
dystrophy,and Lesch-Nyhan syndrome
are relatively rare in most populations,but
because of advances in molecular genetics
they receive attention in the media,
Medical Genetics
More common traits,such as
glucose-6-phosphate dehydrogenase
deficience or color blindness,may occur
frequently enough in some populations to
produce a few affected females,However,
their effect on individuals is rarely life
threatening and medical intervention is
not needed,Pedigree 7 shows one typical
inheritance pattern for a rare X-linked
recessive disease,
Medical Genetics
Medical Genetics
the hallmarks of X-linked recessive
inheritance
(1) As with any X-linked trait,the disease is never
passed from father to son,
(2) Males are much more likely to be affected than
females,If affected males cannot reproduce,only
males will be affected,
(3) All affected males in a family are related
through their mothers,
(4) Trait or disease is typically passed from an
affected grandfather,through his carrier
daughters,to half of his grandsons,
Medical Genetics
Sample pedigree
Medical Genetics
hemophilia
Medical Genetics
Medical Genetics
6,Y-linked
A gene on the Y chromosome,A
Y-linked gene is by necessity passed
from father to son,since the Y
chromosome can only be transmitted
by a man to his male progeny,
Medical Genetics
It has often been said that little is
known about whether specific genes
are or are not Y-linked,
Medical Genetics
a number of genes were known to be Y-linked
including,
ASMTY (acetylserotonin methyltransferase),TSPY
(testis-specific protein),IL3RAY (interleukin-3 receptor),
SRY (sex-determining region),TDF (testis determining
factor),ZFY (zinc finger protein),PRKY (protein kinase,Y-
linked),AMGL (amelogenin),CSF2RY (granulocyte-
macrophage colony-stimulating factor receptor,alpha
subunit on the Y chromosome),ANT3Y (adenine nucleotide
translocator-3 on the Y),AZF2 (azoospermia factor 2),
BPY2 (basic protein on the Y chromosome),AZF1
(azoospermia factor 1),DAZ (deleted in azoospermia),
RBM1 (RNA binding motif protein,Y chromosome,family 1,
member A1),RBM2 (RNA binding motif protein 2) and UTY
(ubiquitously transcribed TPR gene on Y chromosome),
Medical Genetics
Sample pedigree
Medical Genetics
Medical Genetics
7,SPECIAL FEATURES
Penetrance
The likelihood a given gene will
result in disease,For example,if half
(50%) of the people with the
neurofibromatosis (NF) gene have
the disease NF,the penetrance of
the NF gene is 0.5,
Medical Genetics
Expressivity
The consistency of a genetic
disease,For example,Marfan disease
shows variable expressivity,Some
persons with Marfan's merely have
long fingers and toes while others
have the full-blown disease with
dislocation of the lens and dissecting
aneurysm of the aorta,
Medical Genetics
Phenocopy
(1) An environmental condition that
imitates (copies) one produced by a
gene,
(2) The person who has an
environmentally-produced condition
that mimics one produced by a gene,
Medical Genetics
Genetic heterogeneity
Here a similar clinical picture may
be produced by different mutations
at the same locus or at different loci,
Retinitis pigmentosa may be caused
by both autosomal dominant or
recessive inheritance,
Medical Genetics
Anticipation
A remarkable phenomenon in which a genetic
disease appears earlier appearance and with
increased from with each succeeding generation,
Anticipation was once thought not to exist in
genetics,It was chalked off as a meaningless
statistical artifact,However,anticipation has now
been proven to occur in a large number of
important genetic disorders,including Huntington
disease and myotonic dystrophy,In molecular
terms,anticipation is due to the expansion of a
trinucleotide repeat sequence in the DNA,This
phenomenon also occurs in the fragile X
syndrome,the most common inherited form of
mental retardation,
Medical Genetics
Genomic imprinting
The phenomenon of parent-of-origin
gene expression,The expression of a
gene depends upon the parent who
passed on the gene,
Medical Genetics
For instance,two different disorders -
Prader-Willi syndrome and Angelman syndrome -
- are due to deletion of the same part of
chromosome 15,When the deletion involves the
chromosome 15 that came from the father,the
child has Prader-Willi syndrome,but when the
deletion involves the chromosome 15 that came
from the mother,the child has Angelman
syndrome,
Genomic imprinting plays a critical role in
fetal growth and development,Imprinting is
regulated by DNA methylation and chromatin
structure,
Medical Genetics
New mutations
Seen as isolated cases,
Increased paternal age may be
associated with new mutations,
Majority of Achondroplasics are
new mutations,
