Clin Genet

2000: 58: 270 – 279

Printed in Ireland. All rights reser

6ed

Developmental Biology: Frontiers for Clinical Genetics

Collagens: building blocks at the end of the
development line

Section Editor: Roderick R McInnes
e-mail: [e-mail]

Byers PH. Collagens: building blocks at the end of the development
line.
Clin Genet 2000: 58: 270 – 279. © Munksgaard, 2000

Collagens are some of the major building blocks of the vertebrate
body. In addition to their structural role, they are important for cell
guidance during development and for maintaining tissue integrity. In
their absence, phenotypes range from lethal to mild. These studies
demonstrate that collaens,in their rich array, play important roles in
development and are significant elements in reading the developmental
code.

PH Byers

Departments of Pathology and Medicine,
University of Washington, Seattle, WA
98195, USA

Key words: basement membranes – bone

– cartilage – collagen – development –

genes – mutations – procollagen

Corresponding author: PH Byers, Depart-
ment of Pathology, University of Washing-
ton, Seattle, WA 98195, USA. Tel:

+1 206

543 4206; fax:

[phone]; e-mail:

[e-mail]

Received 6 July 2000, revised and ac-
cepted for publication 19 July 2000

Our physical appearance is the end result of the
initiation of a complex developmental program,
the cellular memory of the instructions from that
program, and the cell-, tissue-, and organ-specific
expression of a set of morphogenetic molecules. In
rare instances, the entire morphogenetic program
goes awry presumably because the early program-
ming events in development are miscued (EB
White’s ‘Stuart Little’ being one of the best docu-
mented examples, although still poorly understood
at the molecular level almost half a century after
the first report). Sometimes, these instances of
poor planning lead to rearrangements that can
only be seen with detailed studies of internal
anatomy (such as situs in

6ersus) while others are

more easily appreciated by simple observation (ab-
sence or duplication events, such as forms of ectro-
dactyly or polydactyly). These disorders flow from
fundamental errors in the specification of the body
plan and can be attributed to early molecular
events that occur at specific times in embryonic
development. Although these morphogenetic er-
rors are easy to spot, they are probably the mildest
of the consequences of mutations that affect the
expression or structure of this set of early deter-
mining molecules, the most severe rarely coming to

light because of the great biological efficiency of
our surveillance system for developmental errors.

Collagens: nature’s building blocks

In the real world of dysmorphology, syndromol-
ogy, and genetics there is increasing recognition of
disorders that result from mutations in molecules
that function to organize the developmental role.
At the same time, it is clear that ‘end molecule’
failure leads to some of the more common disor-
ders in which body form is distorted. The distinc-
tive phenotypes resulting from mutations that
affect the amount or structure of type I collagen
(osteogenesis imperfecta [OI] in all its forms) or
type II collagen (with a phenotypic range from
achondrogenesis to Stickler syndrome) are striking
examples (1). Here, the basic plan is laid out, but
the building materials are not delivered appropri-
ately or are unusable because of design flaws.

In this context, the most fundamental of all the

building blocks available are the collagens, until
just the last couple of decades considered to be
some of the least interesting biological molecules
because of their long-term stability. The reputation
of these dynamic, multi-functional molecules was
not enhanced when one of the pioneers of modern

270

Collagen building blocks

molecular biology hoped (perhaps this is apoc-
ryphal) that the structure of DNA would not be
as dull as the triple helix of collagen. ‘Collagen’
has, however, survived and now presents a fasci-
nating tale of evolutionary (almost revolutionary)
adaptation to fill a large number of both general
and highly specialized niches in morphogenesis
and tissue integrity (2).

Most mutations in collagen genes act in a

dominant

manner,

probably

because

the

molecules they form are trimers and incorpora-
tion of a single abnormal chain interferes with
the assembly of three chains in the molecule, the
folding of the triple helix, or interactions with
other molecules in the matrix (3). There are rare
recessively inherited disorders that result from
mutations in fibrillar collagen genes. Recessive
variants of some disorders, dystrophic epidermol-
ysis bullosa that results from mutations in type
VII collagen, can result from homozygosity, or
compound heterozygosity, for functional null alle-
les in collagen genes (4). These experiments of
nature and the deliberate (or fortuitous) inactiva-
tion of collagen genes in model organisms
provide the few instances in which the develop-
mental effects of the absence of these molecules
can be assessed without the interference of the
abnormal molecule.

More than a molecule: collagen is a family of genes
with characteristic features

‘Collagen’ is now known to be encoded by a fam-
ily of at least 30 genes (see Table 1). The protein
products are diverse in size, structure, distribu-
tion, and abundance. Each collagen gene encodes
a precursor chain, known as pro

a chains, of spe-

cified number and type, which contain sequences
that direct them to assemble with specific part-
ners into molecules that contain three chains –
some are homotrimers and some are hetero-
trimers. In aggregate, collagens represent the
most abundant proteins of the body and, for the
most part, function as extracellular building
blocks. Some, notably type XIII and type XVII,
are transmembrane molecules, and others, type
XV and type XVIII, house other functional
molecules – endostatins – that appear to regulate
vasculogenesis and are released from the parent
molecule by proteolysis (5, 6). Thus, it should
come as no surprise that mutations in many of
these genes lead to phenotypes characterized by
altered shape, although not altered form. That is,
the basic instructions for laying down the body
plan are uncompromised; it is only the final con-
struction units that are either present in low

abundance or modified in shape. However, it is
good to keep in mind that some collagens have
other functional components and mutations in
them may have quite different effects on, for ex-
ample, vessel formation.

The biosynthetic pathway is complex

Collagen biosynthesis involves one of the richest
processing pathways of all proteins. Almost all
collagen genes have dozens to more than 100 ex-
ons. The COL

7

A

1

gene has 118 exons, the largest

number of all known vertebrate genes, while the
COL

10

A

1

gene has only two, making it the most

intron poor of all the collagen genes. The fibrillar
collagen genes have between 51 and 66 exons.
The complexity of these genes and the deleterious
consequences of exon-skipping and of the use of
cryptic splice sites provide a rich substrate for
mutations. The mature mRNAs are translated on
membrane-bound polysomes and the precursor
chains are inserted into the lumen of the rough
endoplasmic reticulum. During and after transla-
tion, most prolyl residues that precede glycine
residues in the triple-helical domain are hydroxy-
lated, as are a variable number of lysyl residues,
the number depends on the collagen type. Chains
associate through regions in the carboxyl-terminal
propeptide, which also specifies binding partners,
and triple helix propagates from that end toward
the amino-terminal end of the molecule. Prolyl
4-hydroxylase, a tetramer of two

a-chains and

two

b-chains that are identical to protein disulfide

isomerase (PDI), HSP47 (a collagen-specific chap-
erone), and several other proteins chaperone the
association

and

folding.

Intact

precursor

molecules are secreted, or in the case of type XIII
and type XVII remain membrane-bound through
transmembrane components, are processed prote-
olytically in the extracellular space, and form
fibrillar or meshwork complexes as their final
structure.

Mutations: the common language of dysfunction

For the large part, the phenotypes that result
from mutations in collagen genes reflect the tissue
and organ distribution of the expression of these
genes (Tables 1 – 3). The developmental role of
collagens can be difficult to define when muta-
tions induce additional phenotypes. It is probably
only in the presence of homozygosity for ‘null’
mutations that the role of a particular collagen,
modifying enzyme, or chaperone can be assessed
because of the ability of the organism to adapt to
structural alterations.

271

Byers

Table 1. Collagen genes and their disorders

Gene

Chromosomal lo-

Collagen type

Protein

Disorders

cation

17q21.31-q22.05

pro

a1(I)

Osteogenesis imperfecta

I

COL1A1

Ehlers–Danlos syndrome type VIIA

7q22.1

pro

a2(I)

Osteogenesis imperfecta

COL1A2

Ehlers–Danlos syndrome type VIIB
Ehlers–Danlos syndrome type II

12q13.11-q13.2

pro

a1(II)

II

Stickler syndrome, type I

COL2A1

Wagner syndrome type II
Spondylepiphyseal dysplasia congenita
Kniest dysplasia
Hypochondrogenesis
Achondrogenesis type II
Spondylo-metaphyseal-epiphyseal dysplasia (SMED), Strudwick type

2q31

pro

a1(III)

III

Ehlers–Danlos syndrome type IV

COL3A1

Ehlers–Danlos syndrome type III (?)

13q34

pro

a1(IV)

IV

COL4A1
COL4A2

13q34

pro

a2(IV)

2q36-q37

pro

a3(IV)

COL4A3

Alport syndrome, recessive

COL4A4

2q36-q37

pro

a4(IV)

Alport syndrome, recessive

Xq22

pro

a5(IV)

Alport syndrome, X-linked

COL4A5

Xq22

pro

a6(IV)

Alport syndrome, X-linked Leiomyomatosis

COL4A6

9q34.2-q34.3

pro

a1(V)

Ehlers–Danlos syndrome type I

V

COL5A1

Ehlers–Danlos syndrome type II

COL5A2

2q31

pro

a2(V)

Ehlers–Danlos syndrome type I

Not mapped

pro

a3(V)

COL5A3

COL6A1

21q22.3

pro

a1(VI)

Bethlem myopathy

VI

21q22.3

pro

a2(VI)

Bethlem myopathy

COL6A2

2q37

pro

a3(VI)

Bethlem myopathy

COL6A3

3p21.3

pro

a1(VII)

Epidermolysis bullosa, recessive dystrophic

VII

COL7A1

Epidermolysis bullosa, dominant dystrophic
Epidermolysis bullosa, pretibial

3q12-q13.1

pro

a1(VIII)

VIII

COL8A1
COL8A2

1p34.4-p32.3

pro

a2(VIII)

6q13

pro

a1(IX)

Multiple epiphyseal dysplasia

IX

COL9A1

1p33-p32.2

pro

a2(IX)

COL9A2

Multiple epiphyseal dysplasia, type II

20q13.3

pro

a3(IX)

Multiple epiphyseal dysplasia, type III

COL9A3

6q21-q22.3

pro

a1(X)

X

Metaphyseal chondrodysplasia, Schmid type

COL10A1

Spondylometaphysealdysplasia, Japanese type

1p21

pro

a1(XI)

XI

Stickler syndrome, type III

COL11A1

Marshall syndrome

6p21.3

pro

a2(XI)

Stickler syndrome, type II

COL11A2

Otospondylomegaepiphyseal dysplasia (OSMED)
Weissenbacher-Zweymuller syndrome
Non-syndromic deafness (DFNA13)

6

XII

pro

a1(XII)

COL12A1

10q22

pro

a1(XIII)

XIII

COL13A1

COL14A1

8q23

pro

a1(XIV)

XIV

COL15A1

XV

9q21-q22

pro

a1(XV)

1p34

pro

a1(XVI)

XVI

COL16A1

COL17A1

10q24.3

pro

a1(XVII)

Epidermolysis bullosa, generalized atrophic benign

XVII

21q22.3

pro

a1(XVIII)

XVIII

COL18A1

6q12-q14

pro

a1(XIX)

COL19A1

XIX

272

Collagen building blocks

Table 2. Collagen types, chain composition, and tissue distribution

Chains

Molecules

Collagen type

Tissue distribution

Fibrillar collagens

a1(I), a2(I)

a1(I)

2

a2(I)

I

Ubiquitous in hard and soft tissues, major protein of bone, skin.

a1(I)

3

Uncommon, found in some tumors, amniotic fluid cells

II

a1(II)

a1(II)

3

Cartilage, vitreous, intervertebral disk

See also type XI

III

a1(III)

a1(III)

3

Soft tissues and hollow organs

V

a1(V), a2(V), a3(V)

a1(V)

2

a2(V)

Soft tissues, placental, vessels, chorion

a1(V)a2(V)a3(V)

a2(V) can substitute for the a2(XI) chain in vitreous

See also type XI

a1(XI), a2(XI)

a1(XI)a2(XI)a1(II)

XI

Cartilage

a1(XI)a2(V)a1(II)

Vitreous

Basement membrane collagens

a1(IV), a2(IV)

a1(IV)

2

a2(IV)

IV

Basement membranes

a3(IV), a4(IV)

Others uncertain

a5(IV), a6(IV)

Fibril-associated collagens with interrupted triple helices (FACIT)

a1(IX), a2(IX), a3(IX)

IX

a1(IX),a2(IX)a3(IX)

Cartilage, vitreous

a1(XII)

XII

a1(XII)

3

Soft tissues

a1(XIV)

a1(XIV)

3

XIV

Soft tissues

Meshwork-forming collagens

a1(VIII), a2(VIII)

a1(VIII)

2

a2(VIII)

VIII

Cornea, endothelium

X

a1(X)

a1(X)

3

Hypertrophic zone of the growth plate

Anchoring-fibril collagen

a1(VII)

a1(VII)

3

VII

Anchoring fibrils, dermal epidermal junction

Microfibril-forming collagens

a1(VI), a2(VI), a3(VI)

a1(VI)a2(VI)a3(VI)

Microfibrils in soft tissues and cartilage

VI

Transmembrane collagens

a1(XIII)

XIII

a1(XIII)

3

Cell surfaces, epithelial cells

a1(XVII)

a1(XVII)

3

Epidermal cell surfaces

XVII

Endostatin forming collagens

a1(XV)

XV

a1(XV)

3

Endothelial cells

a1(XVIII)

a1(XVIII)

3

XVIII

Endothelial cells

Others

a1(XVI)

XVI

a1(XVI)

3

Ubiquitous

a1(XIX)

a1(XIX)

3

Ubiquitous

XIX

Type I collagen genes

Type I collagen is the major protein of bone, skin,
tendons and ligaments, blood vessel walls, and
other connective tissues except cartilage. The ma-
jor phenotypes that are known to result from mu-
tations in the COL

1

A

1

and COL

1

A

2

genes are

forms of OI (1), Ehlers – Danlos syndrome (EDS)
type VII (7, 8), an uncommon form of EDS type II
(9), and rare disorders of blood vessel integrity
(10). With rare exception, these disorders are in-
herited in an autosomal dominant fashion and
result either from haploinsufficiency mutations
(11 – 14) or mutations that act in a dominant nega-
tive fashion (1). Survival of individuals ho-
mozygous for ‘null’ mutations in the COL

1

A

2

gene

(moderately severe OI and a rare form of EDS
type II) demonstrate that this is not an essential

gene (9). In contrast, homozygosity for COL

1

A

1

null mutations is lethal in the mouse, which serves
to demonstrate the essential nature of the gene
product (15, 16). These mice die at embryonic day
11 because of loss of integrity of the vascular
system under hydrostatic pressure. With the excep-
tion of some alteration in branching during lung
morphogenesis, the early embryos have no major
form alterations. Their cultured cells do, however,
have multiple alterations of proteins in the extra-
cellular matrix, which may contribute to the lack
of tissue integrity (17).

The COL

1

A

1

gene encodes the pro

a1(I) chains

of type I procollagen, at least two of which are
needed to make a functional molecule. Molecules
cannot accommodate more than a single pro

a2(I)

chain (encoded by the COL

1

A

2

gene). Ho-

mozygosity for COL

1

A

2

non-functional alleles

273

Byers

274

Table

3.

Animal

models,

human

disorders

and

type

of

mutations

in

collagen

genes

Gene

Animal

models

Human

disorders

Type

of

mutation

Type

of

mutation

Homozygous

null

(−/−

)

Heterogyzous

Homozygous

null

(−/−

)

Transgenic

animals

(over

Missense

mutations;

structural

alterations

Heterozygous

null

(+/−

)

null

(+/−

)

expression

or

missense

mutations)

COL1A1

Embryonic

lethal

at

day

11

Wide

range

of

phenotype

from

lethal

to

mild

OI

type

I

OI

type

I

‘like’

Not

seen

Range

from

lethal

to

moderate

in

the

mouse

because

of

phenotype

osteogenesis

imperfecta

vascular

rupture

COL1A2

OI

type

III

Very

mild

os-

Wide

range

of

phenotype

from

lethal

to

mild

NA

OI

type

III,

EDS

type

I/

II

?mild

osteopenia

teopenia

osteogenesis

imperfecta

COL2A1

Neonatal

or

late

fetal

lethal

‘Stickler-like’

Range

from

severe

lethal

to

Wide

range

of

phenotype

from

achondrogene-

Not

seen

Stickler

syndrome

sis

type

II

to

spondyloepiphy-seal

dysplasias

mild

chondrodysplasia

COL3A1

Early

death

from

arterial

rupture

Normal

NA

Not

seen

EDS

type

IV

EDS

type

IV

COL4A1

Point

mutations

are

embryonic

Not

known

Not

known

Not

known

lethal

in

C

.

elegans

COL4A2

Not

known

Not

known

Not

known

COL4A3

Homozygosity

produces

autosomal

recessive

Alport

syndrome

COL4A4

Homozygosity

produces

autosomal

recessive

Alport

syndrome

COL4A5

Hemizygosity

produces

Alport

Hemizygosity

produces

Alport

syndrome

syndrome

COL4A6

Combined

with

loss

of

COL4A5

produces

Alport

syndrome

and

enteric

leiomyomatosis

COL5A1

EDS

type

I/

II

EDS

type

I/

II

COL5A2

Deletion

of

exon

6

(contains

EDS

type

I/

II

N-propeptide

cleavage

site)

produces

EDS

type

I/

II

like

picture

in

homozygous

mice

COL6A1

Early

onset

severe

myopathy

Bethlem

myopathy

COL6A2

Bethlem

myopathy

COL6A3

Bethlem

myopathy

COL7A1

Recessive

dystrophic

Normal

Recessive

dystrophic

epi-

Dominant

dystrophic

epidermolysis

bullosa

Normal

epidermolysis

bullosa

dermolysis

bullosa

COL9A1

Late

onset

mild

degenerative

Multiple

epiphyseal

dysplasia

Normal

joint

disease

COL9A2

Multiple

epiphyseal

dysplasia

COL9A3

Multiple

epiphyseal

dysplasia

COL10A

COL11A1

Perinatal

lethal

in

the

cho

/

cho

Stickler

and

Marshall

syndromes

Normal

Stickler

and

Marshall

syndromes

mouse

COL11A2

Stickler

syndrome

COL17A1

Generalized

benign

epidermolysis

bullosa

Collagen building blocks

produces much milder phenotypes of OI (18) or
EDS (9). Thus, although the COL

1

A

2

gene

product provides some plasticity for the type I
procollagen molecules (particularly, it seems, in
permitting normal bone mineralization and normal
vascular and skin integrity), it is not essential for
survival. In contrast, the absence of COL

1

A

1

gene

products means that no type I procollagen
molecules can be made. Under these circumstances
all mechanical integrity of tissues is lost. This phe-
notype would be expected in a quarter of all preg-
nancies initiated by two parents, both of whom
have OI type I (which results from heterozygosity
for COL

1

A

1

null mutations).

Type II collagen and other collagens expressed in cartilage

Cartilage is home to several collagens: types II, IX,
X, and XI. Type II collagen is the most abundant
collagen in the matrix of cartilage, in the early
anlage of developing bone, and the vitreous of the
eye. Type IX and type XI collagens have similar
patterns of expression and distribution. They form
heterotypic (multiple types of collagen) fibrils with
type II collagen. Because of its appearance early in
the formation of endochondral bone, it seemed
likely that both the form and the growth of the
bones would be dependent on the presence of type
II collagen. In mice homozygous for non-expres-
sion COL

2

A

1

alleles, membranous and periosteal

bone form normally but long bones are rudimen-
tary, lack marrow cavities and the only mineralized
bone is formed from the periosteum. The mice
have no palate; their ribs are small and not prop-
erly mineralized so that the thorax is small. Alveoli
are not distended and the animals do not survive.
Calvarial mineralization is normal, although the
shape is altered (19, 20). In these animals the
notochord is not resorbed and vertebral body
structure is altered (21). It is thought, surprisingly,
that most of these alterations, even the failure of
palate formation and rotation, arise because of
changes in the mechanical properties of tissues, not
specification of the plan of the tissue (21).

Type II collagen is a homotrimer. In addition,

the pro

a1(II) chain is included with the proa1(XI)

and pro

a2(XI) chain in type XI collagen. Muta-

tions that alter sequences in the triple-helical do-
main of the pro

a1(II) chain interfere with the

normal helix formation of 7/8 molecules made by
the cell and many of those alter secretion (1). The
perinatal lethal disorder achondrogenesis type II
results from heterozygosity for mutations in the
triple-helical domain and the phenotype resembles
the homozygous COL

2

A

1

null mouse, probably

because there are virtually no normal molecules

and the abnormal molecules, although poorly
secreted, interfere with fibrillogenesis. The pheno-
type of the heterozygous null mutation in humans,
Stickler syndrome (1), resembles the heterozygous
null mouse.

Type IX and type XI collagens interact with type

II collagen in the cartilage matrix to form het-
erotypic fibrils (that is fibrils that contain all three
molecules). Although both are present in much
lower amounts than type II collagen, the effects of
homozygosity for null mutations (premature termi-
nation codons) in the Col

11

a

1

gene in the cho/cho

mouse are lethal as a consequence of abnormalities
in the cartilage of limbs, ribs, the mandible and
trachea (22). Mutations in the COL

11

A

1

gene in

people give rise to a variant of Stickler syndrome,
Marshall syndrome, or an intermediate phenotype
(23). This variation may depend on the outcome of
splice site mutations. Homozygosity for a null
Col

9

a

1

gene in mice yields a minimal phenotype of

late onset mild degenerative joint disease (24), even
though the protein product is required for assem-
bly of the entire type IX collagen protein (25).

Type X collagen is found almost exclusively in

the hypertrophic zone of the growth plate. Mice
homozygous for inactivation mutants of the
Col

10

a

1

gene have variable phenotypes with about

10% dying in the perinatal period and more by
early adult life. These animals all have significant
growth plate compression as well as hematopoietic
alterations (26). They resemble, to some extent, the
mice produced by a dominant interference trans-
gene (27). In people, dominantly inherited muta-
tions result in Schmid metaphyseal chondro-
dysplasia with short stature and bowed bones. No
homozygous nulls are known but most of the
human mutations may act as heterozygous nulls in
that they likely interfere with chain association or
with multimer formation (28 – 30). Almost all the
mutations in the COL

10

A

1

gene are found in the

domain that encodes the chain association domain.
The relationship between the skeletal and hemato-
poietic findings is not clear at this point, but sug-
gests that the development of the latter may
depend on certain signals from an intact marrow.

Type III collagen

The homozygosity for a Col

3

a

1

null mutation in

the mouse results in a perinatal lethal phenotype
with vascular rupture (31). These mice have very
thin vascular walls and thin skin. As type III
collagen is expressed early in the development of
these tissues, one proposal has been that the
molecules provide a part of the scaffolding on
which the mature organ is built. The published

275

Byers

mutations in the COL

3

A

1

gene all alter the se-

quence of the chains (1, 32). A small number of
premature termination mutations in the COL

3

A

1

gene are known and also result in an EDS type IV
phenotype (vascular, bowel, uterine rupture with
early death), similar to the effects of heterozygous
dominant mutations (U Schwarze and PH Byers,
unpublished).

Type IV collagen

Basement membrane molecules come early and stay
late. They are among the first matrix molecules
synthesized by embryonic cells and the same
molecules are important for isolation of organ-
forming buds from endodermal cells and in the
separation of tissue formed from different cell
pools. These molecules persist in the major base-
ment membranes of the kidney, lung, skin, and
other regions in which cells of one origin are sepa-
rated from those of other origins. In mammals,
there are six type IV collagen genes that occur in
three pairs, each oriented in a head-to-head fashion
and sharing a bifunctional promotor, no doubt
having evolved from a single gene duplication/in-
version event followed by duplication and disper-
sion of the two-gene structure. The two genes
expressed ubiquitously in these tissues are the
COL

4

A

1

and COL

4

A

2

genes, located on chromo-

some 13. No human mutations in these genes are
known and no knockout mice appear to have been
generated. The only clue to the fate of mutations in
these genes is offered in Caenorhabditis elegans. In
C. elegans, mutations in both the COL

4

A

1

and

COL

4

A

2

equivalent genes often have a lethal out-

come during embryogenesis, although others may
permit development to proceed (33 – 35). In hu-
mans, it is likely that most mutations in these genes
result in phenotypes that do not survive early
embryogenesis.

The COL

4

A

3

, COL

4

A

4

, COL

4

A

5

, and COL

4

A

6

genes all appear to be expressed in kidney, either in
heterotrimers that contain a single chain each of the
COL

4

A

3

, COL

4

A

4

, and COL

4

A

5

products or in

trimers that contain two products of the COL

4

A

5

and a single chain encoded by the COL

4

A

6

gene.

Homozygosity for knockout/null mutations in the
murine Col

4

A

3

gene results in a progressive

glomerular disease (36) and cochlear abnormalities
(37), similar to those seen in people with autosomal
recessive Alport syndrome. The most striking effect
of mutations in the type IV collagen genes is the
combined Alport syndrome and diffuse esophageal
leiomyomatosis seen with deletions that extend
from intron 2 of the COL

4

A

6

gene into the

COL

4

A

5

gene (38). It is not clear if there is a gene

involved in the control of smooth muscle growth
located in the large intron 2 or if the presence of
both gene products is important for that function
(39, 40).

Type VI collagen

The type VI collagen genes, COL

6

A

1

, COL

6

A

2

,

and COL

6

A

3

, encode a collagenous protein that

forms a portion of the epimysium and is involved in
connections with many of the proteins of the muscle
fiber. Mutations in any of the three of these genes,
the protein products of which contribute to a single
heterotrimer, result in Bethlem myopathy, an un-
common form of muscle disease characterized by
muscular dystrophy and joint contracture (41 – 43).
Homozygosity for a targeted disruption in the
murine Col

6

a

1

gene results in no clinical phenotype

but histological evidence of myopathy (44). Type VI
collagen is a heterotrimers of all three chains and
the

a1(VI) chain is essential for molecular assembly.

Type VII and type XVII collagens

Mutations in the COL

7

A

1

gene, the encoded

product of which forms anchoring fibrils at the
dermal – epidermal

junction,

produce

different

forms of dystrophic epidermolysis bullosa (45).
Targeted inactivation of the murine Col

7

a

1

gene

results in the recessive dystrophic epidermolysis
bullosa picture, essentially unchanged from that in
humans (46). Mutations in another collagen gene
expressed by basal keratinocytes that functions as a
transmembrane protein, COL

17

A

1

, result in a

much milder form of bullous skin disease (47).

Genotype to phenotype: a well-trodden but poorly
mapped pathway

Dramatic effects on development are seen, to date,
only with null mutations in a small number of
genes: Col

1

a

1

in mice and type IV collagen genes in

C. elegans. Mutations in some of the genes of the
post-translational pathway, notably the prolyl 4-hy-
droxylase gene, may also be an embryonic lethal, at
least when examined in C. elegans (48). It is clear
that, while there are complex interactions among
collagens and other proteins, many of these are
important only at the final stages of growth and
have relatively little importance in the translation of
other developmental signals.

The tissue-specific distribution of gene expression

is one key to understanding the phenotypic effects
of mutations but it remains difficult to see
how mutations that either alter the amount of a
chain synthesized or alter the structure in a very
subtle fashion give rise to the diverse phenotypes

276

Collagen building blocks

represented by dominant interference (dominant
negative) or recessive mutations in collagen genes.
These pathways are most extensively explored for a
few of the fibrillar collagens, type I collagen and
bone disorders, and type II collagen for disorders
of cartilage growth and structure.

The complexity of pathogenesis for mutations in
type I collagen starts in the nucleus

Most known mutations in the COL

1

A

1

and

COL

1

A

2

genes give rise to forms of brittle bone

disease, OI (49, 1). These phenotypes range from a
subtle increase in the risk of fractures (a few during
an individual’s lifetime) to intrauterine or perinatal
lethality (50). The mildest of these forms (OI type
I) results from failure to synthesize the products of
one COL

1

A

1

allele (11 – 14, 51). The mutations

that result in this phenotype are largely premature
termination codons along the length of this 51
exon gene. Three mechanisms are involved: frame
shifts due to small insertions or deletions, point
mutations that create termination codons, and
splice site mutations that result in inclusion of
additional nucleotides or deletion of nucleotides
due to alternate splicing such that new termination
codons result at or downstream from the muta-
tional event. These products are either extremely
unstable or are segregated into the nucleus away
from the protein translation machinery and secre-
tory pathway.

Goes on to the secretory pathway

In the case of mutations that alter the sequence of
the protein, but allow full-length, or nearly full-
length, chains to be synthesized, the cell brings a
very different armamentarium to bear in its attack
on these purveyors of molecular chaos. The consti-
tutive pro

a chains of type I procollagen (and of

other fibrillar collagens) are synthesized on ribo-
somes bound to the membrane of the rough endo-
plasmic

reticulum

and

inserted

through

the

membrane during elongation. Because chain asso-
ciation is determined by sequences in the carboxyl-
terminal 200 residues of these 1400 residue long
chains, the chains lie quietly in the lumen – but
not alone. The modifying prolyl and lysyl hydroxy-
lases, hydroxylysyl glycosidases, and additional
chaperone proteins – probably BiP, GRP78, prolyl
cis – trans isomerase, and HSP47 accompany these
chains in their travels. Once synthesis is completed,
the carboxyl-terminal regions fold, expose interact-
ing surfaces, and chains assemble into appropriate
combinations. By itself, the correct combination of
chains with their mates is a remarkable testament

to the specificity of interactions lent by only a few
residues of a protein backbone. In some cells, the
constituent chains of three or more types of colla-
gen are synthesized simultaneously with few if any
errors occurring in the correct association of the
half a dozen or more different gene products.

Substitutions for glycine, exon-skipping, or

small in-frame deletions or insertions within the
triple-helical domains of these chains do not inter-
fere with association, but do bring helix propaga-
tion to a halt, awaiting sufficient energy to
overcome the barrier or re-initiating helix forma-
tion. These molecules remain bound to the modify-
ing enzymes and their rate of secretion is
substantially slowed. These molecules remain in
the ER for extended periods. Many are degraded
by mechanisms that are not yet clear, but a small
number can still traverse with secretory pathway,
disregarding the many checkpoints on the lookout
for misfolded molecules, only to exit the cell and
continue their misadventures by interfering with
fibrillogenesis or with, in the case of bone, mineral-
ization. The only protection from this rude intru-
sion on order is the intracellular retention and
possibly degradation of the abnormal molecules.
Unfortunately for the organism, these processes
are not entirely successful, so that some abnormal
molecules are secreted and their deleterious effects
prevail.

Mutations that alter sequences in the carboxyl-

terminal region of chains can ablate chain associa-
tion, have no effect, or alter the rate of association.
Those that do not permit chain association act as
‘null’ alleles so that the phenotype would be ex-
pected to be mild. Those that alter the rate of
association activate the cellular ‘stress’ protein re-
sponse and stimulate the synthesis of several chap-
erone proteins which then appear either to
facilitate chain association or interfere with the
process (52 – 54). Although generally inefficient,
some of these molecules are secreted. The pheno-
types tend to be severe, but it is not clear whether
this is true for all such mutations or is biased by
ascertainment.

And ends in the extracellular matrix

Still lacking is a clear step-by-step understanding
of how mutations create the specific phenotype. It
is all well and good to say that too little normal
molecules create a mild phenotype while the pres-
ence of abnormal molecules creates a more severe
picture. After all, although generally true, this is
not always the case. As a rule, for type I collagen
mutations, the phenotype reflects the nature of the
mutation, the location of the mutation in the

277

Byers

molecule, the effect of the mutation on the chain,
and, for substitutions for glycine residues, the nature
of the substituting amino acid. It appears that all
mutations that alter the sequence within the triple-
helical domain of the chain interfere with folding
and, to a greater or lesser extent, decrease secretion.
However, it may well be the secreted molecules that
are the major purveyors of damage. These molecules
are less efficiently incorporated into fibrils and when
present interfere with mineralization. For other
fibrillar collagens, where mineralization is not an
issue, e.g. type III, V, and XI collagens, no doubt
the interaction with other molecular building blocks
disrupts function and structure.

‘Building blocks’ are not the only story

Although perceived largely as the ‘Legos

®

’ of the

skeleton and other tissues, it has become abundantly
clear that collagens have far more diverse functions.
During early development and regeneration of tis-
sue, collagens provide one of the pathways that
direct cell motility and organization. Transmem-
brane molecules, particularly integrins, bind classes
of collagen in a molecular-specific fashion so that
cells can adhere specifically to them. When certain
collagens are not made at all, these interactions
disappear, the ability to produce appropriate cell
guidance and thus to encounter the next set of
signals is lost. These effects are brought to light only
with the ability to inactivate collagen genes, perhaps
because the organism has many adaptive tools
available when structures are altered. Thus, colla-
gens of different types have important roles in
determining cell interactions, cell motility, and tissue
organization. They are important in development,
both from the structural point of view and in
allowing the cells that make them or use them access
to new signal molecules. Mutations in these
molecules that permit survival illustrate the adapt-
ability of the organism and represent the physiolog-
ical response to alterations in a system that has been
tens of thousands of generations in the making.

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