Cover Page

The handle http://hdl.handle.net/1887/28328 holds various files of this Leiden University

dissertation.

Author: Vosse, Esther van de

Title: Positional Cloning in Xp22 : towards the isolation of the gene involved in X-linked
retinoschisis
Issue Date: 1998-01-07
 CHAPTER!

INTRODUCTION
 Introduction

CHAPTER 1 INTRODUCTION

1.1 X chromosome
The X chromosome is about 160Mb in length (1) and contains an estimated 2500-5000 genes.
The X chromosome has many special features that distinguishes it from the autosomes. The most
obvious is that it is one of the sex determining chromosomes; XX individuals are female and XY
individuals are male. All other chromosomes (the autosomes) are always present in two identical
copies but the sex chromosomes differ greatly from each other, not only in size and morphology
but also in gene content. Homologies and differences between the sex chromosomes are
discussed in 1.1.1. Since the X chromosome is present in either one or two copies, unequal
dosage of transcripts of X chromosomal genes in males and females would occur if not X
inactivation would compensate for this. Dosage compensation in XX individuals is provided by
transcriptional inactivation of a large fraction of the genes on one X chromosome; this is
discussed in 1.1.2.

1.1.1 XIY homology

TheY chromosome is 50Mb (2) in length and based on its size could contain an estimated 750-
1500 genes, however, this amount is an overestimation since theY chromosome is gene poor (see
below). TheY chromosome has two main functions: it is required for the male phenotype and
provides a pairing partner for the X chromosome during male meiosis. The gene (or genes)
required for initiating male development is called the testis determining factor (TDF). Only one
gene has been identified that is a candidate for TDF; sex-determining region Y (SRY) (3). Very
few other Y-specific genes have been isolated thus far (2) (the Deleted in Azoospermia gene
cluster (DAZ) gene family (4,5), Spermatogenesis gene on theY (SPGY) (6), testis-specific
protein, Y-encoded (TSPY) gene family (7), Ribonucleic acid binding motifs (RBM) gene family
(8)).
Only a small portion of the genes on the X and Y chromosome are shared, consequently
most genes on the X chromosome are haploid in males. The shared genes are transcribed from
both X and Y chromosome and are located in the pseudoautosomal regions (PAR) which
effectively behave as autosomal genes. The PAR1 region on the Xp and Yp telomeres is a region
of 2.6 Mb (2), delimited by the pseudoautosomal boundary (PAB). The PAR2 region (9) on
subtelomeric Xq and Yq is much smaller (320 kb ). So far only two genes have been cloned from

11
 Cha ter I

this region: the Interleukin 9 Receptor (IL9R) (10) and a Synaptobrevin~like gene (SYBLl) (I I).
The genes identified in PAR I and P AR2 are indicated in Figure 1. In contrast to the genes in
PARI, SYBLl in PAR2 is subject to X inactivation. Even more remarkable is that theY copy
is also inactive. The (in)activation state of IL9R is not known and if this gene shows the same
pattern as SYBLl, a position effect caused by the heterochromatin on Yq may play a role in this.
Another explanation is that these -although located in the PAR- are pseudo-genes.

PAR1
.... ....
Xp22.3 Yp11.3

CSF2RA
IL3A
ANT3
Yq12
ASMT
XE?

MIC2R.:
MIC2 '

V
Xq13

Figure 1: The pseudoautosomal regions.

PARI has a size of2.6 Mb, PAR2 has a size of

.·· ··~ :.
320 kb. The pseudoautosomal regions are
........ SYBL1 / identical in X and Y. The border of PARl is an
Xq28 :.. .. .. .. .. . IL9R •
Alu repeat, the border of PAR2 is a LINE
X PAR2
repeat. XIC localised in Xq 13 is discussed
in 1.1.2.

Pairing between the X and Y chromosome during male meiosis seems to involve only
part of the short arm of the X and Y and includes an obligatory cross-over in PARI. However,
recent reports have been published in which pairing at PAR2 is described (9,12,13). The rate of
recombination in PAR2 is 168 kb/cM (14), in PARI it is 55 kb/cM (15). These rates are very
high compared to the average for the genome (1 Mb/cM), due to the fact that these regions, while

I2
 Introduction

small, have an obligate cross-over (at least in PAR1).

Other regions of homology between X and Y mainly consist of pseudo-genes on the Y,
and are believed to have arisen through non-homologous pairing between the X and Y (16)
followed by inversions (2). These regions are hotspots for illegitimate recombination and are
located in Xp22.3/Yq 11.21, involving recombination between the KAL-X and KAL-Y gene (17),
in Xp22.3/Ypll, involving recombination between PKXl and PKY1 (18), and in Xp22.3/Yp11
in a region just proximal from the PAB, involving recombination between sequences (1/3 in
repeats) that have a high homology (96-98%) between X and Y (19,20).

1.1.2 X inactivation
To compensate for the unequal dose of X genes in males and females, one of the X chromosomes
is inactivated at an early stage of embryogenesis in all somatic tissues in the female. This
phenomenon is called X inactivation or lyonisation after Mary Lyon who first described it in
1961 (21). X inactivation takes place at the time of uterine implantation (22), occurs in all cells
except the germ cells and is random and maintained during all further cell divisions. This
mechanism of dosage compensation is unique to mammals (23).
Basically, all but very few genes on the inactivated chromosome (Xi) are thought to be
inactive except for the genes in PARI and the gene(s) at the X inactivation center (XIC) itself.
On the active chromosome (Xa) all genes are active except for the gene(s) of the X inactivation
center. Amongst the few genes outside PARl that escape X inactivation are for example; SMCX
(24), SB1.8 (25) and ubiquitin-activating enzyme (UBEl) (26). These genes do not have
detectable Y homologues, so their transcription levels are likely to be different in male and
female, the functional significance of this difft;:rence is not yet known. The existence of
functional homologues on the Y chromosome with widely divergent or different sequence can
not be ruled out.
The X inactivation center (XIC) has been localised to Xql3 (Figure 1) (27). X
inactivation spreads from the XIC across the chromosome. The XIST gene (specifying an X-
inactive specific transcript) maps to the XIC and is only transcribed from the Xi (28). X
inactivation is preceded by XIST expressio'l (29) and as no other genes have been found in the
XIC it is assumed that XIST is the gene causing X inactivation. XIST does not code for a protein
and only inactivates the X it is expressed from (in cis). Recent studies using XIST knockout
mice have proven that XIST is essential for X inactivation (30).

13
Chapter 1

The Xi (also known as the Barr body) replicates late in S phase and is visible as
condensed heterochromatin in the nucleus, even in G 1. Although the mechanism is not
completely clear, inactivation seems to be maintained by methylation of the 5' -region of genes
(31). Experiments with patient-derived cell lines with supernumery X chromosomes have shown
that XIC also is involved as part of a counting mechanism to ensure the appropriate activity state
of X-linked genes by allowing only one active X per two sets of autosomes (32).
When one of the X chromosomes harbours a gene that through a mutation is deleterious
to cells in a specific tissue, a skewed X inactivation is observed. This results in the presence of
-in all or most of the cells in this tissue- the non-mutant X as the active chromosome. This is not
caused by a change in the activity state of the X chromosomes but by a selection against the cells
that contain the X chromosome with the mutant gene as the Xa (for example in Incontinentia
Pigmenti (IP) (33)). The opposite effect is often found when larger deletions of the X
chromosome or X/ autosome translocations are present, in those cases the normal X chromosome
is inactivated. X/autosome translocations (34) have been found in, for example, patients with
Duchenne muscular dystrophy (DMD) (35), magnesium-dependent hypocalcemia (HSH) (36),
and Hunter disease (37).

1.1.3 Evolutionary origin of the sex chromosomes

In mammals XX individuals are female and XY individuals are male, but in birds this is the other
way around (38). In the much more distantly related D. melanogaster XX are females and XY
males, while in C. elegans XX are hermaphrodites and XO are males (39). Three different forms
of sex chromosomes are found in fish; some fish have almost identical X and Y chromosomes,
others have an X and Y which hardly recombine and finally there are fish that have lost the Y
completely (38). Obviously, many species have developed sex chromosomes independently
during evolution so there must be a strong evolutionary force pushing all these species to
solutions of similar nature but different in endpoint.
The most likely reason for the initial cosexual species (hermaphrodites) to have favoured
the evolution of separate sexes, is that self-fertilisation is more likely to produce unfit progeny
than sexual reproduction (40). Cosexual species have all genes required for both 'male' and
'female' reproductive organs located on their autosomes. A simple form of acquiring a difference
between sexes is seen in C. elegans, where missing one of these chromosomes causes male
development instead of hermaphroditic. This X-monosomy however causes non-disjunction

14
 Introduction

during meiosis, resulting in non viable embryos in part of the progeny.

In general, X and Y chromosome are believed to have evolved from an autosomal pair
of chromosomes (two 'pre-XY' chromosomes). The first difference between these two
chromosomes may have been the occurrence of a large deletion or inversion on one of them,
disturbing homologous recombination locally. Once homologous recombination was disturbed,
more and more of this mutated (Y) chromosome was lost because it became prone to
rearrangements and steady loss and inactivation of genes (41). In addition, a range of pseudo-
genes originating from autosomal genes have accumulated on the human Y chromosome,
probably through retrotransposition (16). These processes have generated a chromosome which
can only have retained and/or accumulated genes that would enhance male fitness, and will
otherwise only have been selected for appropriate size for efficient meiotic segregation.
Whether the evolution of a dosage compensation system was required before the
degeneration of theY chromosome could start (42) or whether it evolved as a consequence has
not been proven. In C. elegans, transcription of both X's in hermaphrodites is reduced to -50%,
regulated by at least 8 genes and depending on X:A ratio (43). This suggests that dosage
compensation was already available, independent of the presence of a Y chromosome. Dosage
compensation in Drosophila is mainly obtained through increased expression of genes on the
male X (regulated by male-specific lethal genes on the Y), although there is also evidence for a
parallel dosage compensation pathway thit~down regulates some genes on the X in females. In
mammals dosage compensation involves inactivation of most genes on one X in females (see
1.1.2). In many species unique dosage compensation systems have evolved that allowed
development of separate sexes and thereby opened the way to evolution into a 'higher' order of
species.

The mammalian sex chromosomes The size of the X chromosome has been strongly
conserved amongst eutherian ('placental') mammals, being 5% of the haploid genome, and it is
also strongly conserved in gene content (44). Many genes on the human X chromosome have also
been localised to the X chromosome in a wide variety of other eutherian mammals. Comparison
with other therian mammals, the metatheria (marsupials) and prototheria (monotremes), shows
that Xq is part of the X in all therians. Human Xp genes, in contrast, are located autosomally in
both marsupials and monotremes. For instance, in monotremes the human Xp-linked genes
Synapsin 1 (SYN1), DNA polymerase a (POLA) and Ornithine transcarbamylase (OTC) are

15
 Chapter 1

located in one block on chromosome 1, while the Xp-linked genes Dystrophin (DMD),
Synapsin1 (SYN1), Cytochrome b heavy chain (CYBB) and Monoamine Oxidase A (MAOA)
are located in one block on chromosome 2 (45) (Figure 2). In marsupials, MAOA, ZFY, OTC,
DMD, STS, POLA, SYN1 and OATLl have also been shown to be autosomal (46). This means
either that this region was lost from an ancestral X chromosome in the marsupial and monotreme
lineages or was acquired by an ancestral X in the eutherian lineage. Not much is known yet about
the gene content of the Y chromosome in other therians.

~~~A·.::.· ....................... .
ZFX
CYBB
~~~B ·.:·.~·>.;.·:·.::::::::::""""'":::::: ....... . DMD
OTC ·.. \
~~~1A··.·.··:,,.,·.·.:>·.:::,.~:
MAOA

........ ................
··········
:·.: · : .;:~.:.... . OTC
1
SYN1
POLA

2
Figure 2. Assembly of the therian X
············ chromosome. Human Xq genes are found on X
Human X ······ . . . . . . . .
in prototheria as well, human Xp genes are
X present in two blocks on prototheria
Platypus chromosomes 1 and 2.

Not only the gene content of the X chromosome is highly conserved among eutherian
mammals, so is the order. Although blocks of genes have been rearranged, the order of genes
within these blocks is conserved. These rearrangements (through several inversions) are typical
for each subclass. As an example the mouse X is compared to the human X in Figure 3.

16
 Introduction

Xp22.3
Xp22.2
Xp22.1
Xp21.3
Xp21.2
Xp21.1
Xp11.4
Xp11.3

Xp11.2
Xp11.1
Xq11
Xq12
Xq13

Xq21.1
Xq21.2
8
Xq21.3

Xq22 Figure 3: Comparison of the human

Xq23 and mouse X chromosomes. Through
Xq24
Xq25
several inversions in both mouse and
Xq26 human X the order of the genes
Xq27 became different but the 9 conserved
Xq28
blocks can still be recognised. Note:
mouse chromosomes do not show
Human X Mouse X
banding

1.1.4 Deletions in Xp, contiguous deletion syndromes

Deletions and rearrangements of chromosomal regions can greatly facilitate the mapping of
disease genes. Comparison of the deletions or phenotypes in patients with contiguous deletion
syndromes can be used to assign disease genes to a distinct region. For the X chromosome,
deletion mapping has been very useful for the characterisation of several genomic regions, for
example Xp22.3 (47-49) and Xp21 (50-52) (Figure 4). In Xp22.3 amongst others, the
identification of the genes for Kallmann syndrome (53,54) and X-linked ichthyosis (STS)(55)
have been facilitated by the available patients with deletions and contiguous deletion syndromes.
Many of these deletions are thought to be a result of aberrant recombination between the X and
Y chromosome (see 1.2.1). In Xp21, for instance the genes for Duchenne muscular dystrophy
(DMD) (56), McLeod syndrome (XK) (57), and X-linked chronic granulomatous disease
(CYBB) (58) have been identified using deletion mapping.

17
Chapter 1

Xp
··
/
.· ~5Px
MRX
KAL
STS
I interstitial and terminal
deletions found in
males and females
Xp22.3 MLS, AIC, FDH - male lethal
Xp22.2

I
Xp22.1
..... AHC
Xp21.3
interstitial deletions
Xp21.2 DMD
GK found in males and
XK
Xp21.1 CYBB females
RP3
Xp11.4 ..... AIED

Xpi 1.3
Xp11.23
Xp11.22
Xp11.21
Xp11.1

Figure 4. Contiguous deletion syndromes on Xp.

Contiguous deletion syndromes on Xp have been found in Xp22.3 and in Xp2l (extending into Xp11.4).
In Xp22.3 contiguous deletion syndromes are either interstitial or terminal deletions that can involve
combinations of short stature (SS), chondrodysplasia punctata (CDPX), Kallmann syndrome (KAL),
mental retardation (MRX) and X-linked ichthyosis (STS) in both males and females. Microphthalmia with
linear skin defects (MLS), Aicardi syndrome (AIC) and focal dermal hypoplasia (FDH, also known as
Goltz syndrome) are male lethal and therefore almost exclusively found in females. The phenotypes of
these three syndromes overlap so they probably result from a defect in the same gene (64) or are due to a
contiguous deletion syndrome (65). In Xp2l contiguous deletion syndromes are interstitial deletions that
can involve combinations of Duchenne muscular dystrophy (DMD), chronic granulomatous disease
(CYBB), McLeod syndrome (XK), retinitis pigmentosa (RP), mental retardation (MRX, not indicated in
figure since location is still unclear), glycerol kinase deficiency (GK), adrenal hypoplasia congenita (AHC)
and Aland island eye disease (AIED). No contiguous deletion syndromes have been found in Xp22.1-
p22.2.

18
 Introduction

In contrast, in the stretch of DNA between these two regions (Xp22.1-p22.31), deletions
are rare. Deletions found in this region are in general due to inheritance of an X/autosome
translocation and are only found in females where the phenotypic effect is either generated by
spreading of X inactivation onto the autosome, nullisomy of the missing autosomal region, or by
inactivation of the normal X, causing functional nullisomy of the deleted region (35,59). No large
terminal or interstitial deletions (other than through a translocation event) of this region have
been found. The apparent lack of large deletions in the Xp22.1-p22.31 suggests that one or more
genes may be present in this region that, when present in single copy in female, or absent in male,
would be lethal. Consistently, three syndromes in Xp22.31 have been found, microphthalmia
with linear skin defects (MLS), Aicardi syndrome (AIC) and focal dermal hypoplasia (FDH, also
known as Goltz syndrome), that appear to be male lethal.
Mutations in the genes that have been isolated so far from the Xp22.1-p22.2 region are
seldomly due to deletions and when deletions are detected these are small. In the PEX gene,
mutated in X-linked hypophosphatemic rickets (HYP), in only 4 patients out of 150 families
tested, deletions were detected that ranged in size from less than 1 kb to over 55 kb (60). In
PHKA2 (phosphorylase kinase liver a-subunit), the gene mutated in X-linked liver glycogenosis
type I and II (XLG) initial studies showed 1 deletion (of 3 bp) out of 2 XLG I families studied
and 1 deletion (of 3 bp) out of 4 XLG II families studied (61,62). In RSK2, the gene mutated in
Coffin-Lowry syndrome (CLS) an initial screen of76 families revealed two deletions of 118 bp
and -2kb respectively (63).

19
Chapter 1

1.1.5 Disease genes in Xp22.1-p22.2

Several disease genes have been localised in the Xp22.1-p22.2 region (see Figure 5), some of
which were recently found. X-linked glycogenosis type I and II (XLGI and II, MIM 306000) are
caused by mutations in PHKA2 (61,62,66), X-linked hypophosphaternic rickets (HYP, MIM
307800) by mutations in PEX (60), Coffin-Lowry syndrome (CLS, MIM 303600) by mutations
in RSK2 (63). We have focused on RS and KFSD which are discussed below.

X-Iinked juvenile retinoschisis

X-linkedjuvenile retinoschisis (RS, MIM 31270) is an eye disease that causes acuity reduction
and peripheral visual field loss, typically beginning early in life. The first report of what is now
known as RS was by Haas in 1898 (77) who reported the simultaneous findings of changes in
retina and choroid and already suggested hereditary degeneration as possible cause. The term
retinoschisis was introduced by Wilczek in 1935 (78). The first suggestion of sex-linked
inheritance however was by Sorsby in 1951 (79). The frequency is about 1:10.000 (80). Most
patients are diagnosed at school age, although pathological changes are probably already present
at birth and progression is in general slow. Folding and splitting of the macula (simulating cysts)
cause the visual acuity loss (81), intra retinal splitting through the nerve fiber layer causes the
peripheral visual field loss. Severity can range from mild acuity reduction to total blindness at
an early age due to complete retinal detachment (82,83).The RS disease gene has a high
penetrance, with variable expression between families but little variation within a family, this
phenotypic variation may be due to different mutations in one gene. Other explanations for the
phenotypic variation are differences in expression, modifying genes, or environmental factors.
No evidence for genetic heterogeneity has been found (84,85).

Figure 5. Disease gene regions in Xp22.1-p22.2.

The markers and scale (in Mb) are according to the 6'h X Chromosome Workshop (1). Markers are
indicated above the bar, known genes are indicated under the bar. SEDL= spondylo-epiphyseal dysplasia
(MIM 313400) (67), NHS= Nance-Horan syndrome (MIM 302350) (68), RP15= X-linked cone-rod
degeneration (MlM 300029) (69), DFN6= sensorineural deafness (DFN6, MIM 300066) (70), PRTS=
X-linked mental retardation with dystonic movements of the hands (MIM 309510) (71), MRX= non-
specific X-linked mental retardation (MIM 309540) (72-74), RS= X-linkedjuvenile retinoschisis (MIM
312700) (75), KFSD= keratosis fqllicularis spinulosa decalvans (KFSD, MIM 308800) (76), HSH=
hypomagnesemia with hypocalcemia (HSH, MIM 307600)(36).

20

I
 Introduction

TEL
KALIG1 1O
OA1
APXL
CLCN4
PRPS2

Xp22.3

GLRA2
15 ISEDLIHSH
PIGA
MRX19
DFN6
GRPR NHS
Xp22.2 CALB3

IRS
PPEF 20
PHKA2
RSK-2
PDHA1
KFSD
PEX

DXS1226
DXS989
Xp22.1 DXS451 MRX2
SAT RP15
ZFX
PRTS
POLA

DXS1048

MRX38

DXS28

DXS1065

Xp21.3
AHC

GK

DMD
DXS1238

CEN
21
Chapter 1

Except in one case of a female with RS, who was probably a homozygote due to a
consanguineous marriage (86), all reported patients are male. Vision in female carriers is usually
normal. Although publications have stated for a long time that female carriers do not have any
symptoms of the disease (81 ,87 -90) closer examination showed abnormal cone-rod interactions
in some of the carriers (91) and peripheral lesions of the retina in 4 of 5 carriers (92). Features
as reported in these carriers however can also be found in the normal population (93).
The most characteristic clinical finding in RS are macular changes, consisting of any of
the following: splitting, radial folds, pigment dissemination and development of macular scars
(81 ). Other findings include white areas in the peripheral retina, hyperopia, liquefaction of the
vitreous body, vitreous strands, peripheral retinoschisis (in 50% of cases (94)), constricted nasal
r<visual field, subnormal ERG, and a range of rarer fmdings (81).
The biochemical defect of RS is unknown, but histopathologic and electrophysiologic
studies suggest a defect in the Muller cell (93-96) possibly an inability of these cells to remove
the extracellular potassium ions resulting from exposure to light (93,97). In normal eye
development the Miiller cell has a function as a migration determinant for retinal development.
Another theory proposes that the retinoschisis arises from delayed development of the retinal and
choroidal vasculature, causing the retina to outgrow its blood supply (98) but this does not
explain the fovea! schisis. No treatment is available, although surgical intervention is sometimes
performed with varying success rates and often leading to complete retinal detachment and other
complications (83,99,100).

Table I. RS candidate region

Genetic analyses in the RS disease gene region. A= Wieacker et al. 1983 (102), B= Alitalo et al. 1987
(103), C= Gellert et al. 1988 (90), D= Dahl et al. 1988 (90), E= Alitalo et al. 1988 (89), F= Sieving et
al. 1990 (84), G= Alitalo et al. 1991 (104), H= Kaplan et al. 1991(92), I= Oudet et al. 1992(105), K=
Bergen et al. 1993 (106), L= Biancalana et al. 1994 (107), M= George et al. 1994 (85), N= Bergen et
al. 1995 (80), 0= Pawar et al. 1995 (82), P= Shastry et al. 1996 (108), Q= Van de Vosse et al. 1996
(I 09). Marker order is according to the 6th international workshop on X chromosome mapping (110).
* indicates a marker used in lilikage analysis, e indicates the marker with the highest lod score . .A. and
T indicate a recombination between the marker and the RS disease gene. Based on the recombinations
in column M and Q the candidate region for RS is located between DXS418 and DXS999 (hatched
region). The recombinants identified in earlier studies may provide a valuable further refinement of the
region when analysed with markers that have become available more recently between DXS418 and
DXS999.

22
 Introduction

A B c D E F G H I K L M N 0 p Q
Marker
Xg
*
DXS89
* *
DXS143
*
DXS85
* *• * *•• :~

DXS16
* * * * *
>i:T
* * * *
DXS9
*• *•• * *• *
DXS987
* *
DXS207
*•• * *• :f:
*• *•• *•
DXS1053
* •
DXS197
* *•
DXS43
* *• r.*• *•• .''>" .:·· *• .• ,,I;;·* * ·>·,*
. . ·/ .. ·. •. ~
''~;.:'T ;,
··~
~~;,;;.,
"F:'
·~
DXS4;fs/
;: ' ... '. ... ...,,,
I'" :'
,:

1Dxs257 ·,: .,;.. :.· •J: ··;~::~··s 1;'::}\!:! .•. I ~~~: ·i llf' ;. ' <'ti 7
B,xsQ99
: :~:{' :1.:.,. ,:.: . . ·,· ,.. I*
'·i''
~.·..~ l'f' li~ ,: *'~ ~;' '..~~
1..

DXS7161 ·•···.·
* ... ...
DXS443
* * *
DXS1229
*
DXS365
* * ... *
DXS1052
*
DXS274
*••• *••• *•• *
DXS92

DXS1226
* ...
;

DXS41
*• * * *•• *•• *•• * *• .,:k

DXS451
*
ZFX
*
DXS208
*
DXS207
*
DXS28
*
DXS164 *A* *•••
DXS206
*•
DXS84
*•
OTC
*•
23
Chapter I

The first linkage studies and recombination events using RFLP markers placed RS in
Xp22 between DXS43 and DXS41. Further studies using microsatellite markers placed the RS
locus in decreasing intervals (see Table 1), until the present localisation ofRS in a 1Mb interval
between DXS418 and DXS999 (see also Chapter 2).

Candidate genes Recently, several candidate genes for retinoschisis have been cloned by
members of the Retinoschisis Consortium (see note). The frrst two, PPEF (111) and Txp3 (112)
have been excluded as the genes mutated in RS (see Chapter 5). Two others; SCMLI (113) and
Txp7 (114) are still being tested. Recently, the complete RS candidate region has been sequenced
by the Sanger Centre based on clones provided by the Retinoschisis Consortium. Analysis of this
sequence will reveal many novel genes present in the region that can be tested as candidates for
RS.

Note: The Retinoschisis Consortium consists of the following groups:

B.Franco, A. Ballabio in Milan, Italy.
T.Alitalo, A. De la Chapelle in Helsinki, Finland.
D.Trump, J.R.W.Yates in Cambridge, United Kingdom.
W. Berger, H.H. Ropers in Berlin, Germany.
A.A.B. Bergen in Amsterdam, the Netherlands.
T.E. Darga, P.A. Sieving, Michigan, U.S.A.
E. Van de Vosse, J.T. Den Dunnen ln Leiden, the Netherlands.

Note: other forms of hereditary retinoschisis are; autosomal recessive, autosomal dominant and
some unclear hereditary forms of retinoschisis·. Acquired forms of retinoschisis; degenerative
retinoschisis, also called senile retinoschisis and secondary retinoschisis associated with various
diseases, of which diabetic retinopathy is the most common (101).

Keratosis follicularis spinulosa decalvans

Keratosis follicularis spinulosa decalvans (KFSD, MIM 308800) is an extremely rare disorder
affecting skin and eyes. Patients show hyperkeratosis (thickening) of the skin of the neck, ears,
palms and soles, loss of eyebrows, eyelashes and beard, thickening of the eyelids with blepharitis
and ectropion, corneal degeneration, photophobia and baldness (alopecia) in winding streaks. The
symptoms diminish with age. KFSD was first described by Lameris in 1905 (115). The name

24

I
 Introduction

KFSD was given by Siemens (116) and the disorder has also been called Siemens syndrome.
Siemens (117) described KFSD in 1925 as the first dominant sex-linked disease, however, only
about half of the carriers show (mild) clinical symptoms, which is more suggestive of skewed
X inactivation than of KFSD being a dominant sex-linked disease.
Essentially five families have been described thus far, located in Germany (117), the
Netherlands (76), France (118), Finland (119) and the UK (120). Linkage analysis using RFLPs
placed the genefor KFSD in Xp22 between DXS 16 and DXS269 (121 ), analysis of recombinants
using microsatellite markers further refined the region to between DXS7161 and DXS 1226 (76)
(see also Chapter 2). KFSD has been reported to show genetic heterogeneity (122,123) but since
few families are available for research this may just reflect a variation in phenotype between
families.
Since there are so few families with KFSD available, no systematic efforts had been
undertaken yet to specifically clone the KFSD gene prior to this study. However, because the
KFSD candidate region overlaps with other disease candidate regions, several genes have been
cloned that can be tested as KFSD candidate genes purely based on location. The biochemical
defect in KFSD is still unknown.

1.2 Positional cloning of disease genes

To identify the molecular mechanism underlying a hereditary disease, the mutant gene needs to
be identified. If a cellular defect resulting from the mutation is identified, cloning by functional
complementation is possible. If the (defective) protein is known, its identification can lead to the
cloning of the corresponding gene. In most hereditary diseases however, neither protein nor
cellular function are known and in those cases positional cloning is used to identify the disease
gene. In the early days of gene identification, functional cloning was the only way of gene
identification. Since the mid-SO's positional cloning has rapidly taken over simply because
techniques became available that allowed the analysis of larger regions. The ideal approach is
when both functional and positional information can be used to identify a gene.
Positional cloning is usually done following a strategy (illustrated in Fig. 6) that narrows
the search from the complete genome to a small region, preferably a single gene. The first step
is genetic mapping: the region on a chromosome where the disease gene is localised is defined
by linkage and recombinant analysis (discussed in 1.2.1 ). The second step is physical mapping;
identification and isolation of genoinic clones (e.g. YAC, Pl, BAC, cosmid) that are located in

25
 Chapter 1

the disease gene region, construction of contigs and assembly of a restriction map (discussed in
1.2.2). The final stage is isolation of transcripts for candidate genes amongst which the disease
gene may be present (discussed in 1.3).

Linkage analysis Isolation of clones, Digestions, Gene

and recombinants contig construction hybridisations identification

Xp22.2 Noli
Xp22.1

BssHll

Eagl

Sfil

Nrul

X Genetic map Physical map

Slit
~~/..
..
.
······•
•

Restriction map Transcript map

Figure 6: From chromosome to gene.

1.2.1 Genetic mapping

In order to isolate a gene through positional cloning, the genetic location of the gene needs to be
known. Cytogenetically visible chromosomal aberrations, such as translocations or large
deletions, may give a direct indication of the region. When these are not present in the patients,
systematical scanning of the genome with polymorphic markers (linkage analysis) in a subset of
families is sufficient to acquire an approximate chromosomal location. Once an approximate

26
 Introduction

chromosomal localisation has been established, more refined linkage analysis is used to detect
markers close to a disease gene by measuring whether certain marker alleles are statistically more
often inherited together with the disease than the frequency of the allele in the general population
would suggest. The further two markers are apart, the more likely it becomes that a cross-over
between the two markers occurs during meiosis. The general rule for such cross-overs is that
when, amongst every 100 meioses, typically 1 recombination occurs (1% recombination): this
is an interval of 1 centiMorgan (cM). This 1 cM interval on average represents a physical region
of around 1 Mb, but this can differ greatly between regions, due to local differences in
recombination rate along our chromosomes.
In recombinant analysis, usually employed for finer localisation, individual meioses are
analysed to see whether a recombination has taken place between any of the markers and the
disease gene. Identification of recombinations located either distal or proximal to the gene is used
to reduce the candidate region.
The interval to which a disease gene can be localised using genetic mapping is limited
(124). In principle, these limitations are set by the number and distribution of the available
polymorphic markers, the distance (in cM) between these markers (affecting the chance of
detecting cross-overs), the heterozygosity of the markers and the number of available patients.
In practice, however, through the large abundance of genetic markers currently available, the
genetic mapping is mainly limited by the number of patients and hence the number of cross-overs
in the candidate region. The point at which the genetic interval in which the disease gene is
localised is small enough to start physical mapping is hard to define. Starting physical mapping
with a too large genetic interval is a waste of time and energy, while continuing genetic mapping
for too long may not provide the increased refinement of localisation due to lack of informative
recombinants. However, one should always remain alert to new patients and family extension,
i.e. classical advances, since, if a new recombinant appears, this often greatly limits the scan
region.

1.2.2 Physical mapping

Isolation of clones Once a sufficiently small interval has been established by genetic mapping
or by chromosomal aberrations, physical mapping is initiated. One physical method is the
isolation of YAC clones as these contain inserts of -1 Mb, are easy to handle and are readily
available. Markers located in the region can be used to isolate YAC clones either by hybridisation

27
Chapter 1

of gridded YAC libraries (125), PCR screening of YAC pools (126), or -since about 1993- by
screening databases (127). In regions where markers are sparse, chromosome walking can be
done using end-clones (128), jumping and linking libraries (129), Alu-PCR products of
previously isolated YACs (130) or of radiation-hybrids (131). Many whole-genome or
chromosome-specific YAC libraries are available (125,132-134).
Positive genomic clones must be rescreened and their marker content established.
Because a relative high percentage of YAC clones are derived from ligation of DNA fragments
from different genomic regions, chimerism should be checked. This is done either by fluorescent
in situ hybridisation (FISH) of the whole YAC -which will at the same time confirm its
chromosomal localisation-, by mapping YAC end clones using FISH, or by hybridisation to
panels of hybrid cell-lines. The disadvantage of using entire YACs in a FISH experiment is that
small chimeric regions may not be detected. The additional advantage of generating end clones
is that these can be used as markers in subsequent experiments. The length of the YACs is
determined by pulsed-field gel electrophoresis (PFGE).

Contig assembly A contig is assembled based on marker content, on fingerprinting of shared

restriction or PCR fragments or on a combination of the two. The oldest fingerprinting method
is based on comparison of the hybridisation patterns after restriction digestion of the clones and
hybridisation with a repetitive element (e.g. Alu, Line-1, THE) (135-137). The PCR based
methods are based on radioactive PCR using Alu specific or random primers on the clones and
analysing the PCR products after electrophoresis on a sequencing gel (138,139). All approaches
generate a unique pattern of bands for each clone that can be analysed using computer programs.
Because YAC clones frequently show qeletions, rearrangements and chimerism (132, 140)
it is important to analyse several coverages of the whole contig rather than a minimum tiling path
ofYACs. Better still is to have a contig cloned in different cloning systems (e.g. PI-clones and
BACs) to analyse clones from independent sources. An additional advantage of constructing a
contig from different cloning systems is that when certain genomic regions are unclonable or
unstable in one system they may be obtained from another system.

Restriction mapping YACs in general have inserts too large to construct detailed restriction and
transcript maps. A frequently used step to improve the resolution of a contig and to allow the
construction of a restriction map is the isolation of clones that are an order of magnitude smaller

28
 Introduction

than the YAC clones in the original contig. The isolation of smaller clones can be performed by
screening of Pl- (up to 100 kb), BAC- (up to 100 kb) or cosmid (up to 40 kb) libraries (or
subcloning of the YACs into one of these vectors), or by an alternative approach, YAC
fragmentation.

···
A~-,,,,
... ··· . . . insert . ··· ... ... TRP1
llllilllilllll
-11111-----i~f--'';.;.'.;.,'-olllll-olll---~~-~~~~--~~--·..,.·11il0. ..._••__,C::-J!l!?lKI8J"IT!cy~-o;;;;ric_..:;a;.:,:m"-p-1qlllllll5'
TEL URA3 ACEN4 ars1 TEL

pBP108/ADE2
...... amp ori ,~,A ~''""'"'~
~r
TEL HIS3 ADE2 Alu

• 1111 11 c::n;;;$

• 11 Ill r· ~

• Ill Ill ,-~

•
~~---·--____.,
~~~•P-••~•--~m.---~-~
11 11 L_~

~'i>-ltlll-tllll.--lr_~~------1211l4lllll!lllllt

Figure 7: Principle of Y AC fragmentation.

Upon transformation of yeast containing a YAC with plasmid pBP108/ADE2, homologous recombination
between an Alu in the YAC and the Alu in pBP108/ADE2 will occur in part of the yeast cells. Growing
of the yeast on medium lacking tryptophan and adenine allows selection of fragmented YACs (which
contain both ADE2 and TRP 1). A panel of fragmented YACs with various insert sizes is thus generated.

YAC fragmentation Since YAC fragmentation was first described by Pavan et al. in 1990
(141), several improvements of the YAC fragmentation vectors (142,143) made it a rapid and
simple way of generating a panel of clones of decreasing size that can be used for clustering of
markers and clones to defined, consecutive regions ('binning') and restriction mapping. YAC

29
Chapter 1

fragmentation is based on the homologous recombination between a repeat in the YAC insert and
a repeat in a YAC-vector arm containing a selectable marker not present in the original vector
arms. After the recombination the replaced vector arm plus part of the insert is lost and a smaller
YAC is obtained (Figure 7). A panel of fragmented YACs can be used to generate a restriction
map without having to use partial digestions of YACs that are usually difficult to interpret (144 ).
Panels of fragmented YACs have also been used to delimit a duplicated chromosomal region
(145) and to refine translocation breakpoints (146).

1.3 Identification of transcripts

Methods to identify transcripts can roughly be divided in transcript dependent (cDNA based) and
independent (genomic DNA based) techniques. Not one technique is capable of identifying all
genes in a region, so two or more complementary techniques are required to construct a complete
transcription map.
The advantage of cDNA based techniques is that when a cDNA is identified this will
immediately tell something about the tissue and stage it is expressed in and it is proof that the
region is transcribed. The quality of the cDNA is very important, the presence of genomic DNA,
incompletely processed RNA and rRNA should be avoided (by polyA+selection).
The advantage of genomic DNA based techniques is that they are independent of the time
and tissue of transcription, thus enabling the isolation of genes expressed only transiently, in a
specific subset of cells, or at extremely low levels just as well as genes that are expressed
ubiquitously and/or at high levels.

1.3.1 cDNA based gene identification

To identify a gene based on cDNA can be done following three different approaches; screening
of cDNA libraries, cDNA selection and transcript sequencing. The choice of approach mainly
depends on the goal.

Screening cDNA libraries Screening of cDNA libraries is used to identify a specific gene. The
screening is usually done with a specific probe, for instance a genomic fragment deleted in
patients or evolutionary conserved. The technique is simple, as it requires only hybridisation of
a probe to cDNA filters. Many (gridded) cDNA libraries are available and these have been
generated from a range of different tissues and developmental stages. Alternatively, one may

30
 Introduction

generate a new cDNA library from mRNA of any desired tissue. A complementary approach can
be used to identify genes from a more complex source. This involves the hybridisation of
radiolabeled cDNAs (from oligo(dT) primed RNA) to arrays of genomic clones to identify the
clones that contain genes (147) and use those for further analysis.

cDNA selection cDNA selection is the more common approach to isolate transcripts from a
large region and is often used to generate a transcript map from a contig. The first two articles
on cDNA selection were published simultaneously by Lovett et al. ( 148) and Parirnoo et al. ( 149)
in 1991. Several variations have been published since (150,151) but all are based on
hybridisation of cDNA to immobilised DNA, elution, amplification and subsequent rounds of
hybridisation to emich for specifically binding cDNA (Figure 8). The genomic target DNA can
be derived from YAC, P1, BAC and cosmid clones. Clones propagated in bacteria have the

Genomic DNA cDNA

~~
~::;
Immobilise I .
on filter t ' Block
repeats
~~
Hybcidise / ~-~':::::::
'
Elute and amplify
Remove non-specific
cDNAs by washing

specific cDNAs

More amplification rounds Clone cDNA

Figure 8: cDNA selection

31
 Chapter 1

advantage of generating less background than YAC clones. cDNA selection has led to the
· identification of a variety of novel genes amongst which the disease genes for glycerol kinase
deficiency (GKD) (150), hereditary breast and ovarian cancer (BRCAl) (152) and Wiskott-
Aldrich syndrome (WAS) (153).
The disadvantage of cDNA selection is that during the selection not only genuine
transcripts but also pseudo-genes and homologous genes will be isolated that are located in a
different region (usually on another chromosome). On the other hand, these 'artefacts' can be
used to specifically isolate members of a gene family (or the 'parental' gene to a pseudo-gene).
cDNA selection is further limited by the abundance of a transcript in a cDNA library, transcripts
that are present at less than 0.01% are unlikely to be selected for (154).

Transcript sequencing Sequencing random transcripts is not an approach to isolate genes in

a specific region but an approach to isolate all genes present in the genome and one of the major
goals of the Human Genome Project (155). From both the 5' and 3' ends of each transcript a
sequence (200-400 bp) is generated, called an expressed sequence tag (EST). All ESTs are
deposited in a specific database; dbEST, and can thus be screened using sequences from the
region of interest. ESTs that have already been assembled into contigs are present in a separate
database called Unigene. These in silica cloned genes of course need to be verified as to whether
they are derived from the region of interest (and not a homologue on another chromosome) and
whether they are not constructed from two separate genes that happen to share a domain and are
thus 'software-merged' into an overlapping transcript. Recently, many genes have been
identified by in silica cloning, i.e. defined by comparative software analysis, based on homology
to a gene in another species, like the human,thymic shared Ag-1/stem cell Ag-2 gene (TSA-
1/SCA-2) that was identified based on the mouse homologue (156), and two human peroxisome
biogenesis disorder genes (PXR1 and PXAAA1) as the yeast PAS8 and PASS (peroxisome
assembly genes) homologs (157). At least one group has started to systematically compare all
known phenotype-causing genes in one species (D. melanogaster) to human ESTs, in order to
define in silica all homologues and thus to identify potential candidate diseases for these genes
based on their genomic localisation and a potential correspondence of association between
phenotypes of -in this case- Drosophila and human (158).

32
 Introduction

1.3.2 Genomic DNA based gene identification

The four DNA based techniques that can be used to identify genes are evolutionary conservation,
isolation of CpG islands, exon trapping and genomic sequencing.

Evolutionary conservation Functionally important regions in the genome (for instance exons
and regulatory sequences) are conserved through evolution. Thus evolutionary conserved
sequences are likely to be an element of a gene. Evolutionary conservation is detected by
hybridisation of DNA of different species to one another.
Analysis of evolutionary conservation is frequently used to test the presence of a gene
by hybridisation to so-called 'zoo-blots'. Zoo-blots are blots containing DNA from a range of
species, typically DNA of mammals (for instance human, ape, bovine, rodent), birds, fish, etc.
Hybridisation of a genomic fragment to such a blot gives an indication of the extent to which the
fragment is conserved. Although a gene like the DMD-gene was discovered using this approach,
it is a laborious method and is less suitable for analysis of large regions.
A suitable approach for larger scale analyses is the comparison with only one other
species using a protocol similar to cDNA selection. Several rounds of hybridisation and
amplification of genomic DNA from another species to immobilised or biotinylated genomic
DNA of the region of interest will enrich for the conserved sequences (159). Unlike the zoo-blot
method this does not only give an indication of conservation but also provides the homologous
region as an actual clone for further analysis.

[solation of CpG islands A CpG island is a relative short stretch of a G+C rich region (up to
~ kb) in which the frequency of (unmethylated) <;pG nucleotides is significantly higher than
~lsewhere in genomic DNA. About 60% of human genes are associated with CpG islands. They
~re typically located at the 5' -ends of genes in, or close to the promoter region and often include
he first exon of a gene. CpG islands can be identified by restriction enzymes that recognise
tretches of C and G nucleotides and at least one CpG (these restriction enzymes are known as
are cutter enzymes). A CpG island contains a cluster of these restriction sites while normally in
enomic DNA these restriction sites are widely spaced (10s or lOOs ofkb apart). CpG islands are
!so called HTF islands because the enzyme which revealed these sequences, HpaTJ., produced
pall tiny fragments (HTF) (160). There are an estimated 45,000 CpG islands present in the
human genome (161).

33
Chapter 1

Isolation of CpG islands is applicable to both large and small regions and different
techniques can be used. The easiest involves the subcloning of any source DNA using one rare
cutter enzyme and one frequent cutter enzyme and ligating these in a plasmid vector. In this way
a transcription map has been succesfully generated from the Huntington disease gene region
(162) where 24 out of 42 clones contained putative exons and three novel genes were isolated.
A slightly different method involves digestion of the source DNA with a rare cutter enzyme and
ligating linkers to the digested DNA. PCR using one primer directed at the linker and one primer
directed atAlu-repeats will allow the amplification of the CpG islands (163).
A second, more elaborate, approach involves denaturing gradient gel electrophoresis
(DGGE) and is based on the difference in melting temperature between regions with a normal
and a high G+C content. In a denaturing gradient gel, fragments that are G+C rich will melt later
than G+C poor fragments and will therefore have a higher electrophoretic mobility. These faster
fragments can than be isolated from the gel and Cloned. The source DNA needs to be digested
using several enzymes (in this case: Msel, Tsp509I, Nlaiil and Bfal) to provide fragments of
appropriate size prior to DGGE (164).
A third approach involves the digestion of the source DNA with a frequent cutter that
leaves CpG islands in tact (Msel) after which the CpG islands are isolated using a column that
specifically binds methylated DNA (165).

Exon trapping In vivo identification of splice acceptor and splice donor sites, or 'exon trapping'
as it was first described by Auch and Reth in 1990 (166), has been used for both small and large
scale gene identification. Genomic fragments are cloned into a vector containing an exon trap
cassette (a gene preceded by a strong promoter and with a multiple cloning site introduced in one
of its introns) and subsequently transfected into a cell line. Transcription of the ex on trap cassette
gene will incorporate exons present in the genomic fragment, which will then (in principle) be
included in the subsequent splicing (Figure 9). Reverse transcription (RT) and PCR on the RNA
isolated from the cell-line will reveal the trapped exons which can be used for further analysis.
Depending on the vector chosen and RT-PCR protocol applied, one can isolate internal, 3'-
terminal or 5' -terminal exons. Internal exons can be trapped using several vectors (166-171). 3 '-
terminal exons can be trapped using pTAG4 (172). One system allows both internal and 3'-exon
trapping; pETV-SD2 (173,174).
The major disadvantage of exon trapping is that it is sensitive to artefacts: spliced

34
 Introduction

products, involving cryptic splice sites and sequences with fortuitous homology to splice sites,
as well as non-specific polyT/polyT primed products (in 3 '-terminal exon trapping). The exon
trap vectors mentioned above can only contain plasmid size inserts, thereby allowing at best one
or only a few exons to be trapped in one product. Moreover, the loss of the genomic context
through the small insert size gives rise to the isolation of sequences which are recognised as
exons although in nature they are intronic or never even transcribed. Furthermore, the order in
which the trapped exons are present in the genome is lost, which makes further analysis tedious.
Since an average internal exon has a length of 137 bp (175) the products tend to be small and not
especially suitable for screening cDNA libraries or databases.
To overcome these prQblems, two vectors have recently been developed that can contain
larger inserts and will thus allow the simultaneous trapping of multiple exons, leaving the order
intact. The exon trap products generated with these vectors make further analysis easier. These
vectors are the sCOGH-vectors ( 176) allowing the isolation of internal, 3 '-terminal and 5'-
terminal exons and pTAG5 (177) suitable for 3'-terminal exon trapping. The most well known
gene that has been identified using exon trapping is IT15, the Huntington disease gene (178) .

. insert ,insert.
mMT1

r ••..·····~
/ ·· ..

ru ~··...••• r. . . . . . . .
mMT1 / ·· ..
· · · · ·...• ••
GH1 GH2 GH3 GH4GH5 GH1 GH2 ~ ~ ~ GH3 GH4GH5

l Transfection into cells

In vivo transcription
RNA isolation
l Transfection into cells
In vivo transcription
RNA isolation

GH1GH2 GH3toGH5 GH1 to GH5

Exon trap product Empty product

Figure 9. Exon trapping using sCOGH2.

Inserts are cloned in a multiple cloning site in intron 2 of the human growth hormone gene (GH). After
transfection of DNA from the clone into a cell line, in vivo transcription of the GH gene will incorporate
exons from the insert present in the same orientation as the growth hormone gene into the exon trap
product. When no exons (or exons in the wrong orientation) are present in the insert an empty product will
result. mMTl= mouse metallothioneine gene promoter. GHl-5= human growth hormone exons.

35
 Chapter 1

Genomic sequencing The most detailed information of any region is obtained by sequencing
it completely. First one can perform database searches to find homologies with known genes or
ESTs and in addition one can use computer programs to predict the location of genes. Large scale
genomic sequencing has been undertaken to analyse large regions of DNA that are covered with
contigs ( 179) and ultimately the complete human genome will be sequenced as part of the Human
Genome Project.
Before searching databases with long genomic sequences, repeat masking is essential to
prevent the analysis of large series of repetitive sequences. Subsequent database searches can
involve comparison with all known nucleotide or protein sequences, or subsets that are for
instance species-specific, contain only functional motifs, or contain only new sequences (180).
A selection of these database search programs and their application is presented in Table 2. A
range of programs is available to further analyse the output of these searches, many of which
have been adapted for specific projects. Most programs are accessible through email or via the
World Wide Web (WWW) (181).

Further analysis of the sequence can involve a number of computer programs. Programs
predicting exons, open reading frames (ORFs), promotors, and assembling potential genes are
called gene structure prediction programs. An overview of available gene structure prediction
programs is given in Table 3.

Table 2: Database search programs

" = sequences are translated into 6 reading frames before searching.
b =Blitz is also known as SSEARCH or as the Smith-Waterman method.
' = Nucleotide sequence databases: Genbank, dbEST, Unigene, EMBL, DBBJ, HTGS, dbSTS, or a
locally generated database.
d =Protein sequence databases: PIR, SWISS-Prot, GenPept, PDB.
'=Pattern databases: EC pattern, PIMA, PROBMIN, BLOCKS, PRINTS, PIR-ALN, FSSP, PROSITE,
PRODOM, Sbase.
f = Many options are available to search species-specifiC sequences or only new entries.

36
 . -·
Funcf ·-~·-·. .p ''-~ ......... • • 'r-' ... ~ .............................. ..... ...........................................................
Oatab ...Oatab IN
...... " ....................................................

repeat masking XBLAST nucleotide nucleotide REPBASE database

REPEATMASKER nucleotide nucleotide REPEATMASKER database

general database search' FAST A nucleotide nucleotide c. improvement of FASTP

c
BLASTN nucleotide nucleotide

BLASTX translated nucleotide• protein d, post-processing: BEAUTY

BLASTP protein protein d, post-processing: BEAUTY

c
TBLASTN protein translated nucleotide•
c
TBLASTX nucleotide• translated nucleotide•
d
Blitzb protein protein
c
Automat nucleotide/protein nucleotide/protein
c,d
Staden nucleotide/protein nucleotide/protein

search new entries in XREFdb nucleotide nucleotidec monthly automatic search

databases FastAiert nucleotide nucleotidec regular automatic search

e
search pattern database FASTA-PAT protein protein motif
e
for protein motifs FASTASWAP protein protein motif

PROTOMAT protein/nucleotide• protein motif BLOCKS & PROSITE database

e
ProfileScan protein protein motif
e
MOTIFINO protein protein motif

MacPattern protein protein motif PROSITE database

generate 20/30 model of Swiss-Model protein proteins of known structure ExPOB,ExNRL-30 databases ~
protein structure post-processing: ProMod g-
ProteinPredict protein protein SWISS-Prot database f2"
w
--
":::t.
-..]
~

~!'#t:c:tY~3ffr)fw;•J;.:-~-'?-:!;p;,,§&iWWi:"A94M&hi1¥!1£!\1!4k4$W,9iiiiit,,i$W!lk# 1MMM&MM99L.\M;:;;;;::;;;;; &t© 42 .M _;;::;;,; Ji :; .WM& answ; 4#it ;;u;;

 ~
w training set: predictions: sequence length:
00
t'rogram year spec1es genes Spl. UHt" gene aos· max LSnownJ no1es
Testcode 1982 diverse 570d - + - - n.s. .........~
GeneModeler 1990 C. elegans 5 + + + - no (50 kb) integrated approach"·b

Gelfand method 1990 mammalian 9 + + + - n.s. (6.5 kb)

NetGene 1991 human 95 + + - - n.s. i

GRAILI 1991 human 18 - + - - 100 kb better on long (>100 bp) exons !

GeneiD 1992 vertebrate 169 + + + - 20 kb integrated approacha.b,r I

SORFIND 1992 human 116 + + - - 32 kb predicts internal exons only"·' I

Geneparser 1993 human 56 + + - - no predicts internal exons only"·'

Genviewer 1993 vertebrate

GeneMark 1993 prokaryote .. - + - -

SITEVIDEO 1993 human n.s. + - - - n.s. (22 kb)

GREAT 1993 vertebrate ,,

GenLang 1994 diverse 32 + + + - n.s. (20 kb) integrated approach•.b.f

GRAILII 1994 human n.s. + + - - 100 kb output used in GAP31

GAP3 1994 human n.s. - + + - uses output of GRAILII

FGENEH 1994 human 461 + + + - n.s. output is assembled gene onlyb·'

f
Xpound 1994 human + + - -
Geneparser 3 1995 human 59 + + + + no integrated approacha,b,f

PromoterScan 1995 primate 167 - - - - n.s. predicts only polymerasell promoters

f
Gene ID+ 1996 vertebrate + + + + 8 kbe
 Introduction

Table 3: Gene structure prediction programs

"=Integrated approach includes analysis of initiation signal, stopcodon, polyA signal (AATAAA) and
promoter (TATA box).
b = This approach is not especially suitable when more than one gene is present, when overlapping genes
are present or to detect alternative·splicing.
' = Approach especially suitable for partial sequences or when more than one gene is present in the
sequence.
ct =Short sequences, not genes (321 coding, 249 non-coding).
e = May have improved.
r =Used in comparison by Burset et al.(!82) see text.
g = Database searches are used to compare ORF with existing proteins, output not shown.

Extensive comparison of a subset of the gene structure prediction programs (indicated in

Table 3) by Burset and Guig6 (182), showed that 33- 51% of exons are predicted perfect (with
exact splice boundaries), 22- 36% of exons are totally missed, 13 - 27% of predicted exons are
completely wrong. A slightly different evaluation method looks at overlap between actual and
predicted exons, this ranges from 62- 71%. Programs that also predict an amino acid sequence,
generate proteins that show 52- 62% similarity to the actual protein sequence.
The accuracies of the predictions were lower using only new sequences than when using
sequences that were partly available in the databases at the time the programs were trained.
Furthermore, the programs seem to perform worse on long stretches than on short stretches which
will be a problem when large-scale sequence analysis is needed (183).
It is important to realise that the programs bave different, complementary strengths and
the choice of programs depends on emphasis (sensitivity or specificity) and desired features.
However, these programs generate predictions which must be verified as no prediction program
so far is capable of predicting all exons of a gene accurately, and all have a significant false
positive rate. The programs develop rapidly however, and are likely to improve constantly, but
will always stay one or more steps behind of the evolving needs of genomic research.

39
Chapter 1

1.4 Testing candidate genes

Once a gene has been identified it can be tested as a candidate gene for a specific disorder. The
techniques that are available to test a candidate gene are once again complementary, no single
technique can identify all mutations in a given gene. These techniques can be either DNA based
or RNA based. Once it has been proven that mutations in the candidate gene cause the disease,
the same techniques can be used for further mutation analysis. The choice of technique for
mutation analysis depends on the mutation spectrum of the gene, the size of the gene and the
number and size of the exons. The most common techniques are described below, other less
frequently used techniques have been described by R.q.H. Cotton, 1997 (184).

Hybridisation Hybridisation of a gene- or, in an earlier stage of the identification of the gene,
of a genomic fragment- to Southern blots with digested DNA of patients, will reveal genetic
rearrangements caused by deletions, duplications, inversions, or nucleotide changes that alter
restriction sites. Aberrant fragments involving larger genomic regions can best be identified using
pulsed-field gel electrophoresis (185).

Sequencing Sequencing is sometimes chosen, especially for smaller genes, in an initial stage
-when no information is available about the type of mutations to expect- to identify the first
mutations. To reduce the work load, sequencing of a candidate gene in a few patients is usually
performed on RNA-derived material but can also be performed on genomic DNA when a small
gene is involved.

Single Strand Conformation Analysis (SSCA) SSCA is based on the conformational change
of denatured DNA induced by a mutation. In short, DNA fragments (typically 200-400 bp) are
amplified using specific primers in a PCR and these fragments are denatured and run on a
polyacrylamide gel. Missing or smaller products may indicate a deletion of (part of) the fragment
or a mutation at one of the primer sites. The mobility changes are caused by single nucleotide
changes or small deletions. Since analysis of the full length of the genomic DNA of a given gene
is laborious, SSCA primers are usually designed in intron sequences flanking exons to amplify
specifically the latter. A major disadvantage of this is that many mutations in introns that alter
splice sites will be missed (186). Also, rearrangements will be missed in which the exons are

40
 Introduction

present, but no longer in the right place or orientation. This was found for the factor VIII gene,
which is inverted in a large fraction(> 40%) of severe hemophilia A patients (187).

Denaturing Gradient Gel Electrophoresis (DGGE) In short, (double-stranded) PCR products

are separated on a polyacrylamide gel with an increasing temperature or an increasing
concentration of denaturant (urea/formarnide). When the temperature or concentration of
denaturant in the gel has been reached at which the low-temperature melting domain will become
single-stranded, the electrophoretic mobility of the product is greatly reduced. The precise
conditions in which this happens and thus the precise position in the gel are highly dependent on
the specific nucleotide sequence. Any change in this by a mutation is likely to cause an altered
migration. Due to the PCR-process, when generating the fragments in heterozygous samples,
besides the normal and mutant strands also heteroduplexes are generated. These are even less
stable and their gel position tends to differ even between the heteroduplexes with normal and
mutant sequence in the two different strands. However, only mutations in the low-temperature
melting domain of a PCR product can be detected. In order to analyse the original high-
temperature domain as a low-temperature domain, a new high-temperature domain is created
by addition of a 'GC-clamp' (by a GC-rich tail on one of the primers) that will alter the melting
characteristics of the product (194). Single base mutations and small deletions will cause an
increase or decrease in the melting temperature that can be detected as a product that runs higher
or lower in the gel than the wild type product.

RT-PCR and protein truncation test The Protein Truncation Test (PTT) (188) is a technique
based on the analysis of the encoded protein. cDNA is generated by reverse transcription (RT)
of patient-derived RNA The cDNA is amplified using PCR to generate stretches of 1-2 kb. One
of the primers used for the PCR contains a transcription initiation signal and a T7 promotor ( 189)
to facilitate transcription and the subsequent translation (Figure 10). The first check for
aberrations is done by running the PCR products on an agarose gel.
In vitro translated products, analysed on SDS/PAGE gel, reveal mutations that directly
or indirectly (through a frame-shift) cause a premature translation termination. This technique
has been successfully used to identify a large number of mutations in hereditary cancers, e.g. the
gene for hereditary breast and ovarian cancer (BRCA1) (190) and adenomatous polyposis coli
(APC) (191). Also, PTT has allowed to find the first protein truncating mutation in CBP in

41
Cha ter 1

Rubinstein-Taybi syndrome (RTS) patients and thus to unambiguously implicate CBP in causing
RTS (192).
One of the requirements for the application of this technique is that the gene is transcribed
in the tissue that is used for RNA isolation (usually lymphocytes). However, illegitimate
transcription (193) of many genes normally not expressed in lymphocytes often provide enough
transcripts to produce a PCR product.

A B
RNA

• •
RNA ---·····~······
Reverse
transcription
In frame deletion
DNA Protein-.. . _. . .

~ • PCR (one primer

with T7-tail)
RNA

~
7;,_

• Protein • Stop mutation

• Transcription

•
mRNA RNA

•
AUG

Translation Frame shift

Protein Protein

Figure 10. PTT principle.

A. RNA is reverse transcribed into cDNA. The cDNA is then amplified using a primer containing a T7 -tail
and a translation initiation signal. In vitro transcription and translation results in a protein that can be
analysed on a SDS/PAGE gel. B. In frame deletions in the RNA will result in a decreased protein size,
the decrease proportional to the size of the deletion. Stop mutations will result in a smaller protein, the size
of the protein depending on the position of the stop mutation. Frame shift mutations usually result in a
premature stop downstream thus also generating a smaller protein.

42
 Discussion

1.5 DISCUSSION

Since the beginning of the 90s, major improvements have been made to existing gene
identification techniques but no revolutionary new techniques have been developed to identify
genes in the human genome. Most groups are using a combination of the available techniques,
usually including either ex on trapping or cDNA selection and gratefully make use of the rapidly
expanding EST database, which is of enormous assistance in the isolation of disease genes (195).
New genes are found and published every week. In 1996, in the monthly journal Nature Genetics
alone, up to 12 new genes were published per month (in total 105), on average 6-7 monthly.
The Human Genome Project (HGP), started in February 1988 with the National Research
Council (NRC) report: 'Mapping and Sequencing of the Human Genome' (155). The three main
objectives for the years 1990-2005 of the HGP were: 1) to improve the research infrastructure
of human genetics, 2) to help establish DNA sequence as the primary interface between
knowledge of human biology and knowledge of the biology of model organisms 3) to launch an
open-ended effort to improve the analytical biochemistry of DNA.
To reach these goals the HGP aimed to develop genetic and physical maps of the human
genome and to sequence the human genome by the year 2005. In addition genetic and physical
maps of mice, worm, flies and yeast would be developed as these are valuable model organisms
for studying development, diseases and treatments. In the early years there was considerable
skepticism about whether the available technology would be adequate. Technical advances
however, especially in PCR, FISH and YAC cloning, have been so great that the speed of genetic
and physical mapping has rapidly gone up (196).
Since the start of the HGP, many human genome maps have been published based on
genetic or physical data or an integration of both (127,137,197-204). Systematic sequencing of
contigs has already generated many megabases of human sequence (44 Mb at 28/2/97, 170 Mb
predicted at 28/2/98 (205)) and it will not be long before the complete sequence of the human
genome will be available. That is not however, the end of the human genome analysis, but merely
a step along the way toward understanding of the genes and their functions.
In conclusion, the time when the genome sequence of many organisms will be publicly
available to boost biological research, is not far away. In a few years therefore, in what tends to
be called the 'post-genome era', the focus of many groups will transcend from building contigs
and transcript maps to the (large scale) functional analysis of the genes that have been identified

43
Chapter I

in silica. Developmental expression patterns, differentially spliced products, protein folding and
processing, protein-protein interactions, biochemical pathways and regulatory networks need to
be analysed.

The impact of these developments is already fundamentally altering positional cloning.

Genome sequence based approaches will more and more replace parallel and serial transcript
mapping on constructed contigs. Thus, the elucidation of the gene causing RS seems not far off.
While two novel RS candidate genes have recently been cloned 'the old way', meanwhile, using
a clone contig provided by the Retinoschisis Consortium, the candidate region for RS has
recently been rapidly sequenced by the Sanger Centre (Hinxton, UK) and made available on the
internet, thus providing the kick-off for using novel in silica approaches. So it seems only a
matter of time to prove which of the genes cloned or predicted in the region is mutated in RS
patients.
The candidate region for KFSD has not been sequenced yet. Several genes in the region
have been cloned that have not even been tested yet as candidates. The identification of the
KFSD gene will take longer primarily because it will be harder to prove which gene causes the
disorder since only so few families are available .
Once found, the functional analysis of the RS and KFSD genes will lead to an
understanding of the mechanisms underlying these diseases. As RS only involves the eye, there
is good hope for a potential therapy once developed, as the retina may be more easily accessible
for delivery of normal genes through viral vectors (206). KFSD is mainly a skin disorder, which
also makes it more easily accessible than the organs affected in several other severe genetic
diseases, like skeletal muscle in muscul¥ dystrophies or the immune system in
immunodeficiencies. However, much biological research is still required and it is even possible
that the outcome of the genome-based research will allow the development of pharmacological
rather than genetic means of intervention.

44
 Outline

1.6 OUTLINE OF THE THESIS

In this study we aimed at the positional cloning of disease genes in Xp22.1-p22.2. To this end
we constructed a YAC contig covering this region including the markers from DXS414 to
DXS451. The contig enabled us to order new markers in the region and to refine the localisation
of the genes for X-linked juvenile retinoschisis (RS) and for keratosis follicularis spinulosa
decalvans (KFSD) (Chapter 2.1). One of the key YACs in the RS candidate region was used in
YAC fragmentation experiments to generate a panel of fragmented YACs used for 'binning'
clones and to construct a 2.5 Mb restriction map ofthis region (Chapter 2.2).
To identify candidate genes for the diseases localised in this region we have applied ex on
trapping. Initially, exon trapping was performed with cosmids subcloned in the vector pSPL3,
isolating -among others- exons from a known gene: the liver a-subunit of phosphorylase kinase
(PHKA2). To analyse larger genomic regions more efficiently, we have designed a new ex on trap
vector (sCOGH2) which allows the direct analysis of cosmid-size clones (Chapter 3). We have
subcloned key YACs from the region into the sCOGH vectors and used these in ex on trap
experiments. We have isolated several novel transcripts from the region, of which one was
analysed in detail (Chapter 4.1).
Two genes, PPEF and Txp3, which have been isolated by members of the Retinoschisis
Consortium were tested as candidate genes for RS (Chapter 5) but no mutations could be detected
thus far.

45
Chapter 1

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