Osteogenesis Imperfecta

Osteogenesis imperfecta (OI) (“brittle bone disease”)
is a heterogeneous group of clinically and
genetically different types of diseases with a
total frequency of at least 1 in 10 000 individuals.
Spontaneously occurring bone fractures,
bone deformity, small stature, defective dentition
(dentinogenesis imperfecta), hearing impairment
due to faulty formation of the auditory
ossicles, and blue sclerae (the fully
developed conjunctiva of the eye is thinner than
normal, so that refracted light is shifted toward
blue) occur in the various forms to different extents
and with different grades of severity, depending
on the type of mutation.

Molecular mechanisms in osteogenesis imperfecta

Some types of mutation may lead to reduced
production of pro!1(I) (1 and 2), e.g., deletion of
a COL1A1 allele, a transcription or splicing defect,
or faulty formation of collagen fibrils. The
relative excess of pro!2(I) molecules becomes
degraded. Thus, less procollagen than normal is
formed, but it is not defective. Numerous other
types of mutations can lead to defective procollagen
(3). Mutations in the pro!1(I) gene are
more severe than mutations in the pro!2(I)
gene because a greater amount of defective collagen
is formed.

Mutations and phenotype

The location of a mutation in the gene influences
the phenotype. Generally, mutations
in the 3! region aremore serious than mutations
in the 5! region (position effect). Mutations of
the pro!1(I) chain are more severe than those
in the pro!2(I) chain (chain effect). The substitution
of a larger amino acid for glycine, which
is indispensable for the formation of the triple
helix, leads to severe disorders (size effect).
Different types of mutations may occur, such as
deletions, mutations in the promoter or enhancer,
and splicing mutations. The codons
(AAG, AAA) for the amino acid lysine, which occurs
frequently in collagen, are readily transformed
into a stop codon by substitution of the
first adenine by a thymine (TAG or TAA), so that
a short, unstable procollagen is formed.

Different forms of osteogenesis imperfecta

Osteogenesis imperfecta may be classified according
to severity into four basic phenotypes
(Sillence classification). Although the classification
does not correspond to the types of mutation,
it has in general proved clinically useful. Ol
types I and IV are less severe than type II (lethal
in infancy) and type III. Three radiographs show
a relatively mild (but for the patient nevertheless
very disabling) deformity of the tibia and
fibula in Ol type IV (1); severe deformities in the
tibia and fibula in Ol type III (2); and the distinctly
thickened and shortened long bones in
the lethal Ol type II (3). Mutations in Ol are
autosomal dominant, the severe forms being
due to de novo mutations. Germline mosaicism
has been shown to account for rare instances of
affected siblings being born to unaffected
parents.

Molecular Basis of Bone Development

The skeleton develops from mesodermal cells
committed to differentiate into three specialized
cell types: chondrocytes (cartilage-forming
cells), obsteoblasts (bone-forming cells),
osteoclasts (bone-degrading cells), and their
precursor cells. Osteoblasts produce most of the
proteins for the extracellular bone matrix and
control its mineralization. The osteoblast cell
lineage involves osteoblast-specific transcription
factors (OSFs). One such transcription factor
was identified in 1997 as a major regulator
of osteoblast differentiation, the core-binding
factor Cbfal.

The mouse Cbfal transcription

The mouse Cbfal transcription factor (the
human counterpart is referred to as CBFA1) is a
member of the runt-domain family. The runtdomain
is a DNA-binding domain homologous
to that produced by the Drosophila pair-rule
gene runt.

Effects of homozygous Cbfa1 mutations on the mouse skeleton

Targeted disruption of the Cbfa1 gene in the homozygous
state (!/!) results in a severe phenotype.
Homozygous mutant mice are small and
die from respiratory failure at birth. In contrast
to normal mice (+/+, 1), the mutant mice (!/!, 2)
completely lack bone development, as shown
by lack of alizarin red staining. Just before birth,
normal +/+ mouse embryos (3) show welldeveloped
bones (stained red) in the upper extremities,
including the clavicles (arrow) and
the tuberositas humeri (circle). Heterozygous
mice (+/!, 4) show severe hypoplasia and reduced
ossification of the long bones. Homozygous
mutant mice (!/!, 5) totally lack any bone
formation, as shown by lack of red staining.

Mice heterozygous for a mutation in the Cbfa1 gene

Mice heterozygous (+/! ) for a mutation in the
Cbfa1 gene on mouse chromosome 17 showlack
of ossification of the skull (2) compared with
normal mice (1). Normal calcified bone is
stained red by alizarin red, here at embryonic
day 17.5, three and a half days before birth. Cartilage
is stained blue by alcian blue. Heterozygous
mice lack a clavicle (4, arrows) in contrast
to normal mice (3).

Cleidocranial dysplasia in humans

Cleidocranial dysplasia (CCD,McKusick 118980)
is an autosomal dominant skeletal disease
characterizedmainly by absence of the clavicles
and deficient bone formation of the skull. CCD is
now considered a generalized bone dysplasia.
Radiological findings are consistent with generalized
underossification (Mundlos, 1999).
Patients can oppose their shoulders (1) due to
absence of the clavicles (2; photograph by Dr. J.
Warkany, Cincinnati). The calvarium (skull
case) is enlarged with a poorly ossified midfrontal
area (3).

The human CBFA1 gene

The CBFA1 gene, at chromosome location 6p21,
encodes a transcription factor of the core-binding
factor (CBF) family. The human CBFA1 gene
has two alternative transcription initiation sites
with two promoters (P1 and P2) and seven
exons. Part of exon 1 and exons 2 and 3 encode
the DNA-binding runt domain; exons 4, 5, 6, and
7 code for transcriptional activation and repression
domains. The nuclear localization signal
(NLS) is located at the 5" end of exon 3. Exon 6 is
alternatively spliced and unique to CBFA1. The
role of the CBFA1 gene also includes amajor regulatory
function in chondrocyte differentiation
during endochrondral bone formation (Mundlos,
1999). As such, it functions as a “master
gene” in bone development.

Mammalian Sex Determination and Differentiation

Sex Determination
In the 1940’s, the French embryologist Alfred
Jost observed that when the undifferentiated
gonads were removed from a male rabbit fetus
before male development had begun, it
developed as a female. In 1959, chromosomal
analysis of two disorders in man, Turner syndrome
and Klinefelter syndrome, yielded the
first evidence that genetic factors on the Y chromosomes
of mammals are important in determining
male sex. A specific gene on the mammalian
Y chromosome (SRY, sex-related Y) induces
male sex development during embryogenesis
(sex determination).

Determination of male phenotype by the Y chromosome

Individuals with Turner syndrome have only
one X chromosome (no Y chromosome) and a
female phenotype, although incompletely
developed and usually accompanied bymalformations.
Individuals with Klinefelter syndrome
have two X chromosomes, a Y chromosome,
and a male phenotype, although also incompletely
developed

Sex-determining region SRY on the Y chromosome

The relevant region in man lies in the distal
short arm of the Y chromosome at Yp11.32. The
short arm and the proximal half of the long arm
of the Y chromosome have been divided into
seven intervals (2). The most distal region of the
short arm is the pseudoautosomal region 1 (PAR
1). This region is homologous to the distal segment
of the short arm of the X chromosome.
Homologous pairing occurs here with crossingover
during meiosis. The physical map of the
pseudoautosomal region and the proximal half
of interval 1 (1A1–1B) span somewhat more
than 2500 kb in man (3). Intervals 2–7 contain
no genes for male sex determination. The crucial
portion of the Y chromosome for male sex
determination in man is about 35 kb (in the
mouse about 14 kb) of a region designated Sry
(sex-related Y chromosomal region)

Male development of an XX mouse transgenic for the Sry gene

Clinical observations and experimental evidence
indicate that the presence of SRY induces
male development, irrespective of the presence
of the remainder of the Y chromosome. A chromosomally
female transgenic mouse (XX)
shows normal male development after the 14
kb DNA fragment carrying the Sry region of a
mouse Y chromosome is implanted into its
blastocyst.

Sry expression

Sry expression during embryonic
gonadal development of the mouse
During embryonic development of an XY
mouse, Sry is expressed only between days 10.5
and 12.5. The subsequent events leading to
male development are initiated during this
short time of expression.

Sex Differentiation

Sex differentiation (development of a given sex)
consists of many genetically regulated, hierarchical
developmental steps. In mammals, the
development of male structures requires induction
by appropriate genes.

Indifferent anlagen of sex differentiation

The gonads (1), the efferent ducts (mesonephric
and paramesonephric) (2), and the external
genitalia (3) all develop from an indifferent
stage. At about the end of the sixth week of
pregnancy in humans, after the primordial
germ cells of the embryo have migrated into the
initially undifferentiated gonads, an inner portion
(medulla) and an outer portion (cortex) of
the gonads can be distinguished. When a normal
Y chromosome is present, early embryonic
testes develop at about the 10th week of pregnancy
under the influence of a testis-determining
factor (TDF). If a normal Y or TDF (SRY) is not
present, ovaries develop. The wolffian ducts,
the precursors of the male efferent ducts (vas
deferens, seminal vesicles, and prostate),
develop under the influence of testosterone, a
male steroid hormone formed in the fetal testis.
At the same time, the müllerian ducts—precursors
of the fallopian tubes, the uterus, and the
upper vagina—are suppressed by a hormone,
the Müllerian Inhibition Factor (MIF; also
known as anti-müllerian hormone, AMH).
When testosterone is absent or ineffective, the
wolffian ducts degenerate. The müllerian ducts
develop under the influence of estradiol, a hormone
produced by the fetal ovaries. The external
genitalia (3) in humans do not develop until
relatively late, starting in the 15th to 16thweek.
Full development of male external genitalia depends
on a derivative of male-inducing testosterone,
5-dihydrotestosterone, a metabolite of
testosterone produced by the enzymatic action
of 5!-reductase.

Sequence of events in sex differentiation

Sex differentiation proceeds in a cascadelike
manner, with a series of temporally regulated
successive steps at different levels of differentiation.
After the primordial germ cells migrate
into the undifferentiated gonads, early embryonic
testes develop under the influence of
testis-determining factor (TDF) if a Y chromosome
is present. TDF is identical with the Yspecific
sequences of the SRY region (see
p. 386). During normal male differentiation, the
further development of the müllerian ducts is
suppressed by the müllerian inhibitor factor.
Testosterone can exert its effect only in the
presence of an appropriate intracellular receptor
(androgen receptor TFM, see p. 390).
When a Y chromosome is not present or when
the SRY region is missing or altered by mutation,
testes are not formed. In this case the
wolffian ducts cease to develop. In the absence
of testes, ovaries develop from the undifferentiated
gonads; the wolffian ducts degenerate;
and the müllerian ducts differentiate into
uterine tubes, uterus, and the upper vagina.
Testosterone also has an effect on the central
nervous system (“brain imprinting”). It is assumed
that this is required for the psychosexual
orientation apparent later in life. When testosterone
is absent or ineffective due to a receptor
defect, gender orientation is female.
In the majority of genetically determined disorders
of sexual differentiation, gonadal and
genital sex do not correspond (pseudohermaphroditism).
In true hermaphroditism,
where the gonads consist of both testicular and
ovarian tissues, male and female structures
exist side by side.