Group Title: Cerebrospinal Fluid Research 2005, 2:2
Title: Genetic loci for ventricular dilatation in the LEW/Jms rat with fetal-onset hydrocephalus are influenced by gender and genetic background
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Title: Genetic loci for ventricular dilatation in the LEW/Jms rat with fetal-onset hydrocephalus are influenced by gender and genetic background
Series Title: Cerebrospinal Fluid Research 2005, 2:2
Physical Description: Archival
Creator: Jones HC
Totten CF
Mayorga DA
Yue M
Carter BJ
Publication Date: 38515
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Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
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Cerebrospinal Fluid Research Bioed Central



Research

Genetic loci for ventricular dilatation in the LEW/Jms rat with
fetal-onset hydrocephalus are influenced by gender and genetic
background
Hazel C Jones*1,2, Crystal F Totten', David A Mayorgal, Mei Yuel and
Barbara J Carter1


Address: 'Department of Pharmacology and Therapeutics, University of Florida, Gainesville, FL 32610, USA and 2Dr. H. C. Jones, Gagle Brook
House, Chesterton, Bicester, Oxon OX26 1UF, UK
Email: Hazel C Jones* hjones@ufl.edu; Crystal F Totten pebblscftuf@yahoo.com; David A Mayorga David.Mayorga@mail.house.gov;
Mei Yue meiyues@hotmail.com; Barbara J Carter bcarter@biotech.ufl.org
* Corresponding author



Published: 12June 2005 Received: 16 December 2004
Cerebrospinal Fluid Research 2005, 2:2 doi: 10.1 186/1743-8454-2-2 Accepted: 12 June 2005
This article is available from: http://www.cerebrospinalfluidresearch.com/content/2/1/2
2005 Jones et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: The LEW/Jms rat strain has inherited hydrocephalus, with more males affected than
females and an overall expression rate of 28%. This study aimed to determine chromosomal
positions for genetic loci causing the hydrocephalus.
Methods: An F, backcross was made to the parental LEW/Jms strain from a cross with non-
hydrocephalic Fischer 344 rats. BCI rats were generated for two specific crosses: the first with a
male LEW/Jms rat as parent and grandparent, [(F x L) x L], designated B group, and the second
with a female LEW/Jms rat as the parent and grandparent [L x (L x F)], designated C group. All
hydrocephalic and a similar number of non-hydrocephalic rats from these two groups were
genotyped with microsatellite markers and the data was analyzed separately for each sex by
MAPMAKER.
Results: The frequency of hydrocephalus was not significantly different between the two groups
(18.2 and 19.9 %), but there was a significant excess of males in the B group. The mean severity of
hydrocephalus, measured as the ventricle-to-brain width ratio, was ranked as B group < C group
< LEW/Jms. For the both rat groups, there were several chromosomes that showed possible
regions with association between phenotype and genotype significant at the 5% or 1.0% level, but
none of these had significant LOD scores. For the C group with a female LEW/Jms parent, there
was a fully significant locus on Chr2 with a LOD score of 3.81 that was associated almost
exclusively with male rats. Both groups showed possible linkage on Chrl7 and the data combined
produced a LOD score of 2.71, between suggestive and full significance. This locus was largely
associated with male rats with a LEW/Jms male parent.
Conclusion: Phenotypic expression of hydrocephalus in Lew/Jms, although not X-linked, has a
strong male bias. One, and possibly two chromosomal regions are associated with the
hydrocephalus.





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Introduction
Fetal hydrocephalus occurs in humans from causes such
as intraventricular hemorrhage and intrauterine infec-
tions, but in other cases the cause cannot be identified
with certainty. Epidemiological studies provide evidence
that hydrocephalus has a genetic component [1-3],
although only one inherited form, X-linked hydrocepha-
lus, has been characterized at the molecular level [4].
Rodent hydrocephalus mutants have been known for
many years [5] and a few mouse mutants have been genet-
ically characterized [6-8]. The publication of the first DNA
assembly for the rat has enabled the rat genome to be inte-
grated with DNA sequences from other species [9]. It is
now possible to identify homologous regions between rat
and human or rat and mouse, and to place disease-related
genes from the human or mouse on the rat genome. Addi-
tionally, the identification of candidate genes for specific
traits in rats is possible through comparative mapping.
The study of disease-related genes in the rat will lead to a
better understanding of inherited conditions in humans.

The LEW/Jms rat was first described in 1983 as being
derived from an inbred strain of Wistar-Lewis rats and as
having lethal fetal-onset hydrocephalus with a frequency
that varied between litters from 12 25% and sometimes
higher [10]. A six-generation pedigree showed that about
25% of the breeding pairs did not produce hydrocephalic
pups. The authors concluded that there was a Mendelian
autosomal recessive mode of inheritance.

Seven rats of the strain were received at the University of
Florida in year 2000 and their DNA was tested with 87
selected microsatellite markers. All but two of these mark-
ers were both homozygous and homogeneous indicating
that the strain was almost completely inbred [11]. Since
2000 the strain has been maintained by brother-sister
mating and almost all successful breeding pairs have pro-
duced hydrocephalic offspring. This suggests that hydro-
cephalus may not be a Mendelian recessive trait [11].
Severe hydrocephalus is evident soon after birth from a
domed head, with death occurring soon after weaning.
Therefore the strain is maintained by breeding from
apparently non-hydrocephalic rats because pups with
overt disease do not survive to reproduce. It was found,
however, that some adult ex-breeding rats have a milder
form of hydrocephalus. These rats, however, did not pro-
duce pups with an increased frequency for hydrocephalus,
which would have been expected with direct transmission
of the trait. The overall frequency of hydrocephalus
among pups was 27.7%, with a significant excess of
affected males. Crossing to another rat strain, Fischer 344,
produced a small number of pups with hydrocephalus
(3%). A backcross from the F1 progeny to the LEW/Jms
strain produced hydrocephalic pups, also with an excess
of males and a frequency of hydrocephalus of 18.8% [ 11].


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The presence of affected pups in the F1 generation and the
high frequency of affected BC1 pups suggest that the trait
may be semi dominant and controlled by one or possibly
two genetic loci. This study aimed to perform a genome-
wide scan and QTL analysis on backcross progeny to iden-
tify chromosomal regions) associated with the hydro-
cephalus. Using gender-specific crosses, the genotyping
has revealed one and possibly two loci associated with
hydrocephalus.

Materials and Methods
Animals
For all experiments the 'Principles of Laboratory Animal
Care' (NIH publication no. 86-33, revised 1985) was fol-
lowed. All rats were pathogen free at the start of the exper-
iment and were housed for the duration of the experiment
in a single room under conventional conditions. Patho-
gen monitoring was performed periodically. The LEW/
Jms strain was donated by Dr. K. Sudoh, University of
Tokyo, to H.C.J. at King's College, London, UK, in 1987.
Between 1991 and 2000, they were housed at the Univer-
sity of Manchester Institute of Science and Technology,
Manchester, UK (C. S. Bannister). Seven animals trans-
ferred to the University of Florida in 2000 were the
founder rats for the current breeding colony and for this
backcross experiment. The animals used in this study were
selected at random from the four breeding lines described
previously [11]. Inbred Fischer 344 rats were purchased
from Harlan (Harlan F344/Hsd). This strain does not
develop hydrocephalus and was used in a previous genetic
analysis with the H-Tx hydrocephalic strain [12].

Breeding
In the first part of the study, the LEW/Jms (L) rats were
bred to Fischer 344 (F) and the F1 progeny backcrossed to
LEW/Jms as described previously producing 1574 back-
cross (BC1) progeny [11]. A genotype analysis using the
complete set of BC1 progeny did not produce meaningful
results. Hence the results were examined according to the
sex of the parents (see Results, Analysis of genotypes). Of
1574, 599 had LEW/Jms as the paternal parent for both
generations, designated 'B' group [(F x L) x L], 114 of
which had hydrocephalus. A further 365 were designated
'C' group with LEW/Jms as the maternal parent for both
generations [L x (L x F)] and 68 had hydrocephalus. Addi-
tional backcross breeding was carried out to increase the
number of rats within these two specific crosses. For the
'B' group progeny, 8 female F1 rats bred from LEW/Jms
males and F344 females, were crossed with 8 male LEW/
Jms rats and 373 BC1 progeny were generated. For the 'C'
group progeny, 24 male F1 rats bred from F344 males and
LEW/Jms females, were crossed with 24 LEW/Jms females
and 608 BC1 progeny were generated. Records of the
breeding pairs and litters bom were entered into a



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database (Filemaker Pro, Filemaker Inc, CA, USA) for
both LEW/Jms breeding colony and the backcross.

Analysis of phenotype
In a previous study it was shown that the severity of
hydrocephalus as measured by the ratio of ventricle-to-
brain width is independent of age between pups aged 2-
23 days after birth. This was true for both the LEW/Jms
colony and the BC1 rats [11]. To measure phenotype rats
were euthanized with CO2 suffocation, or in the case of
pups less than 10 days old, with an overdose of sodium
pentobarbital (100 mg/kg). The brains were excised, fixed
in 10% neutral buffered formalin and sliced coronally at
1 mm thickness using a fine blade. The slices were exam-
ined and photographed under a binocular microscope. A
slice at the level of the striatum was photographed and the
dilatation measured as the ratio of ventricle width-to-
brain width (hydrocephalus severity, [13]). Non-hydro-
cephalic (control) rats were given a nominal phenotype of
0.01 because although ventricles are not visible on 1 mm
slices, small ventricles are found in histological sections
[14]. Phenotype measurements were entered into the
MAPMAKER program for analysis. The data was com-
pared to data from the brains of 392 rats from the LEW/
Jms parental strain described previously [11].

Genotyping
With the exceptions described below, BC1 progeny were
sacrificed between 2 and 23 days of age. Liver tissue was
removed, frozen in liquid N2 and stored at -80 C for DNA
extraction. Because it was found previously that a propor-
tion of ex-breeding LEW/Jms rats had ventricular dilata-
tion when examined post mortem [11], some BC1 rats were
raised until 22-24 weeks of age, at which stage they were
sacrificed and tissues removed; n = 54 rats from five litters
for the B group and n = 60 rats from five litters for the C
group. Genomic DNA was extracted from liver tissue
using the standard chloroform-phenol method, amplified
by PCR using primers for microsatellite markers (Rat Map
Pairs, Research Genetics or Invitrogen) and separated by
agarose gel electrophoresis as described previously [15].

For the initial study, DNA was extracted from all 247
hydrocephalic rats and from 168 littermates that had no
ventricular dilatation (non-hydrocephalic rats). This set of
BC1 rats had mixed parentage, consisting of 29% that had
a female LEW/Jms parent for the first generation and a
male for the backcross, 39% with both LEW/Jms parents
being male rats (designated 'B' type), 23% with a female
LEW/Jms parent in the first cross and also in the backcross
(designated 'C' type), and 8.5% with a female parent in
the first cross and a male for the backcross. Genotyping
was performed in stages using a panel of 96 genome-wide
microsatellite markers. The mean spacing between mark-
ers was 14.36 cM and the largest was 41.64 cM on Chr20,


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where informative markers were scarce. Apart from this,
two other markers on Chrs7 and 18 had spacings >30 cM
(30.40 and 31.75) and all other spacings were < 30 cM.
The results were examined with the X2 test for significant
departure from the null hypothesis that the ratio of
homozygous to heterozygous genotypes was 50:50.

For the additional rats generated in the B and C categories,
DNA was extracted from the frozen liver of all overtly
hydrocephalic pups an equal number of non-hydro-
cephalic littermates, and from pups found to have mild
hydrocephalus after examination of the brains. First-stage
genotyping was performed using DNA from 30 or more
hydrocephalic and non-hydrocephalic rats from each
group, with the same genome-wide panel of 96 microsat-
ellite markers. The data was combined with data from the
initial study, analyzed separately for each group, and
examined for significance at the 5% level. The presence of
significance at the 5% level, while not sufficient for likely
linkage, was used as a guideline to determine the strategy
for possible further genotyping. Genotyping was then
continued on specific chromosomes where there was sig-
nificant association, until all rats had been included.
Additional markers were included to increase the density
on these chromosomes. The data for each chromosome
was analyzed by MAPMAKER.EXP to determine the best
marker order and by MAPMAKER.QTL to calculate the
LOD score [16]. Significance levels were determined using
a LOD score of 1.9 (P < 0.0034) for suggestive significance
and a score of 3.3 (P <0 .0001) for full significance as
defined by Lander & Krugylak [17].

X Chromosome analysis
Since there was an excess of males with hydrocephalus, a
possible association with ChrX was sought. Genotypes of
the B type progeny with a male LEW/Jms parent at each
generation, were informative for ChrX. The C progeny
could not be used because the contribution from the non-
hydrocephalic F344 strain came from ChrY. Seven micro-
satellite markers polymorphic for the LEW/Jms x F344
cross were genotyped on all 'B' rats and the data analyzed
independently for each sex by the X2 test.

Human-rat homology to search for candidate genes
Candidate genes were sought for the chromosomal
regions where there was significant evidence for a hydro-
cephalus locus as determined by MAPMAKER. A strategy
was used similar to that described previously for the H-Tx
rat linkage analysis [12]. The Ensemble Rat Genome
Browser, a joint project between the European Molecular
Biology Laboratory-European Bioinformatics Institute
and the Sanger Institute (http://www.ensembl.org, ver-
sion 26.3 d.1, 08/02/2004) was used to identify the meg-
abase (Mb) positions for the microsatellite markers in the
vicinity of the hydrocephalus loci. The likely genetic


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Cerebrospinal Fluid Research 2005, 2:2


positions for the loci were identified based on the LOD
score maps generated by MAPMAKER. Possible candidate
genes were selected from known rat genes and from pre-
dicted genes that were identified from homologous
regions on the human and the mouse genomes. Genes
were then evaluated as potential candidates using a
number of different criteria [12].

Results
Breeding and expression of hydrocephalus
LEW/Jms parental strain: From breeding records kept over
a period of 3.5 y, the overall frequency of overt hydro-
cephalus was 28% out of a total of 2401 pups (Fig. la, col-
umn 'all'). As reported previously, there were significantly
more males with hydrocephalus than females, X2 = 46.21,
but no significant difference between the sexes of non-
hydrocephalic rats. Instead there was a small, but signifi-
cant excess of total males over total females, X2 = 12.3 (Fig
Ib). The frequency of hydrocephalus varied with parity, in
that the percentage in second and third litters, 31.2% and
47.6% respectively, was significantly increased over that
in first litters, 22.2%, P < 0.001, X2 test (Fig la). Although
the frequency in the fourth and fifth litters was also
increased over the first litter, there was no statistical signif-
icance, possibly because the numbers were small as some
females did not continue to breed after the third litter. The
average litter size decreased from 9.5 pups in first litters to
5.0 in fifth litters.

Backcross progeny
Table 1 shows the total number of BC1 pups sacrificed at
2-23 days of age and numbers genotyped for each group.
There was an excess of males over females with hydro-
cephalus in the B group, X2 = 15.57 (Table 1, Fig. Ib). The
same trend was also present in the C group, but not signif-
icant. Both groups also had an excess of female over male
non-hydrocephalic rats, although again, it was only signif-
icant for the B group, X2 = 11.02. The frequency of hydro-
cephalus in the B and C backcross progeny was not
significantly different between the two groups, 18.2% and
19.9%, respectively (Figs. Ic and Id, column 'All'). Simi-
lar to the parental LEW/Jms strain, the frequency of hydro-
cephalic pups depended on parity and was significantly
lower in the first litters than in subsequent litters. This
effect was much less evident for the B progeny than for C
progeny, which had the female LEW/Jms parent (Figs. Ic
and Id). The ratio of male to female hydrocephalic pups
was not significantly different between the 1st, 2nd, 3rd and
4th litters for either group, X2 test. Among the backcross
progeny sacrificed at 22-24 weeks, there were seven rats
with mild hydrocephalus in the B group (n = 54) and nine
in the C group (n = 60). An additional two had severe, but
non-fatal disease in the B group and three in the C group.
As with the pups, there was also an excess of males with


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hydrocephalus in these two groups. However, the sample
was small and there was no statistical significance.

Analysis of Phenotypes
The mean severity of ventricular dilatation for the B group
was 0.58 +/- 0.01 and for the C group was 0.61 +/- 0.01.
This difference was significant, P < 0.05, Kruskal-Wallis
test. There was no significant difference between the mean
severity for males and females in either group (data not
shown). Dilatation severity was 0.66 +/- 0.01 for the LEW/
Jms parental strain, which was significantly higher than
for B or C rats, P < 0.01 and 0.05, respectively. As reported
previously, there was no significant difference in hydro-
cephalus severity between males and females in the paren-
tal strain [11].

Analysis of genotypes
The genotypes for the first backcross progeny with rats
from mixed mating groups were examined using X2 test for
association between phenotype and genotype. On Chrl 7
at marker D17Ratl7, there was significance at the level of
P < 0.05. No other marker on any chromosome had a sig-
nificant result. The data was re-tested after separation of
the genotypes into four groups according to sex of the
parental rats. However, as already stated, the number of
rats was too low at this stage for the results to be meaning-
ful. The genotypes obtained for B and C rats in the initial
study were combined with data for the additional B and C
rats bred subsequently. First, the genotypes were analyzed
for the male and female data combined. In addition,
because of the strong male bias in the expression of hydro-
cephalus, the data was analyzed separately for males and
for females (Tables 2 and 3).

B group QTL mapping
Analysis of the data for male and female rats combined
showed two or more markers on each of four chromo-
somes with significance at the 5% level or above by X2 test
(Chrs 1, 5, 17, and 19). QTL analysis was performed with
MAPMAKER for these chromosomes using the combined
data, and also on data for each sex separately. None of the
chromosomes reached the level required for full signifi-
cance (3.3) for both sexes combined, but a score sugges-
tive for significance was achieved on Chr5, LOD = 1.94,
and almost achieved on 19, LOD = 1.89 (Fig 2). Separate
male and female MAPMAKER analyses for chromosome 5
indicated a greater level of significance for females than
males (Table 2, Figs. 2a, 2b). For chromosome 19, there
was a higher significance level for the males (Table 2, Fig.
2c) and the contribution of the females to the combined
LOD score was very small (Fig. 2d). On Chrl7, the peak
LOD for both sexes was 1.84 at D17Rat65 and again, the
effect was largely on the males (Table 2). The X chromo-
some marker DXRat83 gave a significant result, P < 0.05
for the male rats in this B group (Table 2). In addition, the


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a Effect of parity on hydrocephalus
expression: LEW/Jms rats


2 3 4 5 All
Litter Number


Effect of parity for [(F x L) x L],
B Group
50-

M 40-
0
2 so. 22

20 16 4 18.2%
194
S10 2

0


1 2 3 4
Litter Number


5 All


Ratio of males to females


ml Non-hydrocephalic
Hydrocephalic
SAll
ns
ns ns ns ns




LEW/Jms BC1 'B' BC1 'C'



Effect of parity for [L x (L X F)],
C Group


' -

S4 *** 4


34
o 3 199

o- 36

0- H -- -- H


1 2 3 4
Litter Number


5 All


Figure I
a: Percentage of hydrocephalus in LEW/Jms rats by litter number (parity) and total percentage (All). There was a significant
increase in percentage of affected rats between the ISt and 2nd and the ISt and 3rd litters, P < 0.00 1. This was not maintained for
the 4th and 5th litters. The number of litters in the data sets is depicted above the columns.b: The ratio of males to females in
the parental LEW/Jms strain and in the BC, progeny. In the parental strain, there were significantly more males than females
with hydrocephalus (black bars, P < 0.001), and also among the total pups born (striped bars). In the B group [(F x L) x L],
there was a significant excess of females among the non-hydrocephalic pups, (open bars, P < 0.001 and a significant excess of
males in the hydrocephalic pups, P < 0.001. In the C group [L x (L x F)], there were no significant departures from the
expected 1:1 sex ratios for the hydrocephalic or the non-hydrocephalic rats (ns).c: Percentage of hydrocephalus in [(F x L) x L]
BC, rats by litter number (parity) and in total. There was a significant increase in the percentage expressing hydrocephalus
between the Ist and 2nd litters, P < 0.001. This was not maintained in subsequent litters. The number of litters in the data sets is
depicted above the columns. d: Percentage of hydrocephalus in [L x (L x F)] BC, rats by litter number (parity) and in total.
There was a steady and significant increase in expression between Ist litters and all subsequent litters, P < 0.001. The number
of litters in the data sets is depicted above the columns.


data showed that chromosomes 11 (for males) and 13
(for females) had results for one or two markers signifi-
cant at the 5% level or higher (Table 2). The low signifi-
cance levels and small LOD scores obtained for this group


of rats did not contribute meaningful results to this
genetic analysis. The exception was data for Chrl 7, where
the results were combined for the B and C groups (see
below).


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Table I: Numbers of BC, pups of each phenotype bred and genotyped (in parenthesis). In the B group there were more males than
females with hydrocephalus, P < 0.001 (a) and more females than males in the non-hydrocephalic group P < 0.001 (b). The differences
were not significant for the C group.

Parentage Sex Hydrocephalic Non-hydrocephalic Total

B Group [(F x L) x L] Male 109 (108)a 330 (91) 439(198)
Female 58 (58) 421 (51)b 479(109)
C Group [L x (L x F)] Male 102 (102) 342 (82) 444 (184)
Female 80(79) 386 (58) 466 (137)




Table 2: Genotypes for B type [(F x L) x L] male and female rats showing markers for which the association between phenotype and
genotype was significant at a level of 0.05 (*) or 0.01 (**).

[(F x L) x L] Males Genotypes

Hydrocephalic Non-hydro-cephalic
LL LF LL LF P2 P value
Chrl DO I Rat56 45 63 51 40 4.09 *

DO I Rat57 46 62 43 27 6.03 *

DO I Rat65 47 60 52 37 4.09 *

DOIRat219 40 60 52 39 5.61 *

DO I Rat67 47 59 54 38 4.06 *

DO IRat208 46 62 52 39 4.18 *

Chr 5 D05Rat49 45 33 26 38 4.10 *

Chr II DI Rat28 50 42 32 54 5.25 *
DI Rat73 53 40 35 56 6.33 *

Chrl7 D17Rat85 65 40 40 51 6.31 *
D17Rat65 68 39 40 50 7.21 **

Chrl9 D19Rat28 68 40 43 52 6.39 *
D19Ratl2 66 41 42 52 5.82 *
D19Rat40 68 39 44 45 3.95 *
D19Rat95 63 43 32 41 4.22 *

L F L F x2 P value

ChrX DXRat83 60 43 39 51 4.28 *

(F x L) x L] Females LL LF LL LF 2 P value

Chrl DO I Rat36 24 34 31 19 4.57 *

Chr5 D05Rat36 35 23 19 32 5.79 *
D05Rat41 38 18 24 27 4.74 *

Chrl3 D13Rat85 19 28 32 18 5.40 *

Chrl9 D19Rat28 36 24 22 33 4.59 *






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Table 3: Genotypes for C type [L x (L x F)] male and female rats showing markers for which the association between phenotype and
genotype was significant at a level of 0.05 (*) or 0.01 (**).


Genotype


[L x (L x F)] Males


Hydrocephalic


Non-hydro-cephalic


D02Ratl5
D02Rat34
D02Rat91
D02Rat52
D02Rat49
D02Rat46
D02Rat24
D02Rat62
D02Rat65
D02Rat250

D04Ratl 12
D04Rat72

DIORat28

DI6Rat81

D 7Arb3
D17Arbl3
DI7Ratl81
DI7Ratl7


[L x (L x F)] Females


D02Rat21

D02Ratl35
D02Rat26
D02Rat34
D02Rat91
D02Rat46
D02Rat52
D02Rat49

D07Rat195

D 7Arb4
DI7Ratl81
DI7Ratl7
D 7Arb5


C group QTL mapping
The combined analysis for male and female C rats showed
quite different results to that seen for the B group. Instead,
chromosomes 2 and 4 were significant for two or more
markers at the 5% level or above. Of these, chromosome
2 had a peak of LOD = 3.81, indicating a locus with full
significance for hydrocephalus situated near D2Rat241
(Figs 3a,b). The LOD score for chromosome 4 (1.59) did


not reach the level for suggestive significance. Similar to
the B group, there was a peak on Chrl7 at a different loca-
tion, D17Ratl3, LOD = 1.73, but it was not significant.
The males and females were analyzed separately (Table 3).
The locus on Chr2 was gender specific in that it had a
much larger effect on males with a LOD score of 3.43,
whereas for the females the LOD score was only 1.41
(Table 3, Figs. 3a,b). The effect on Chr4 was almost totally


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Chr2













Chr4



ChrlO

Chrl6

Chrl7


P value

*
**
*
*
*
*
**
**
*
**

**
*


Chr2


Chr7

Chrl7


*
*
*
*


P value

**

**
*
*
**
**
**
**








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Chr5: male B [(Fx L)x L)]


Male + Female 1.94
-----------------------1'


I --- .- 60 100 120 140 160
n I . .& . .L*"


Chr5: female B [(Fx L) x L)]


160
-j cM


D5Rat120 D5Rat132 D5Rat141
D5Rat92


I I I
D5 6 D5Rat49
D5Rat93


Chr19: male B [(FxL)xL]


D19Rat28


D19Rat40


Chr19: female B [(Fx L)x L]


2 Male + Female 1.89
"%--- ------------


1, 0 0 60 70
. . . . . . . . _h A


D19Rat12


DlRat95 1 Dl9Rat64
D1Mg~at64
DMgRat2l
DMgRat9O


Figure 2
LOD score graphs created from the MAPMAKER output for [(F x L) x L], B rats on chromosomes 5 and 19. The X-axes rep-
resent the recombination distance in centi-Morgans (cM). The microsatellite markers are positioned on the X-axis and named
below. The horizontal dotted line represents the score required for suggestive significance (1.9). a: Plot for male B rats on Chr
5 and b: plot for female B rats (solid lines), dotted line represents a portion of the plot for both sexes combined, where the
LOD score (1.94) reached suggestive significance. There was a strong female bias (3b). c: Plot for male B rats on Chr 19 and d:
plot for female rats (solid lines). The dotted line near the centromeric end represents part of the plot for both sexes combined
where the LOD score (1.89) is very close to suggestive significance for linkage. The male rats contributed to this peak almost
exclusively.


on the male rats (Table 3). For Chrl7, males and females
were affected equally (Table 3). In addition to chromo-
somes 2, 4, and 17, described above, chromosomes 10
and 16 (for males) and 7 (for females) had data for one or
more markers significant at the 5% level or above (Table
3). Only Chr2 was studied further, apart from Chrl7
where the data was combined for both groups (see
below).


B and C QTL combined
All data sets were combined for QTL analysis of Chrl7.
The maximum LOD score for all rats was 2.71, between
suggestive and full significance, and situated near
D17Rat62. For males it was 2.07, also close to D17Rat62,
but for females the maximum LOD was only 1.26 and sit-
uated in a different position at D17Arb5 (Figs. 4a,b).


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c


2-


o
0
S 1-
0
_j
O


Cerebrospinal Fluid Research 2005, 2:2









Cerebrospinal Fluid Research 2005, 2:2


Chr2: male C [Lx(LxF)]


http://www.cerebrospinalfluidresearch. com/content/2/1/2


Chr2: female C [Lx(LxF)]


ao,
002
,


ee + Ferrde 3.81


D2Rat116 / 1V 1 /2at D~4 D15 DT263

D2 Rat11D2R173 t D2Rat52 D2Rt62
D2Rat152


Figure 3
LOD score graphs created from the MAPMAKER output for [L x (L x F)] C rats on Chr 2. The X-axes represent the recom-
bination distance in centi-Morgans (cM). a: plot for male rats and b: for female rats (solid line). The horizontal dotted line rep-
resents the score required for suggestive significance (1.9) and the solid line the score for full significance (3.3). The two graphs
are quite different for males and females with the males reaching a score indicative of full significance (3.43) in the same loca-
tion as the map for both sexes combined (dotted line, LOD = 3.81).


Chr17: combined B & C male
rats


Male + Female 2 71






10 2 30 40 50 60 70


I /
D17Rat59 D17Arb3 \ D 17Arb5
D17Arb13 I \ 7
D17Rat181 D17Rat
D17Arb4


4-

3-



2-
1.


M


\ \ D1 7R2at65
D17RatS9


Chr17: combined B & C female
rats


Male + Female 2 71


10 2 30 40 50 60


70


[ / 1 D b R* D67Rat65
D17Rat59 D17Ra 8tl D 17Arb5 D17Rat62
D17Rat118 D17Rat89


Figure 4
LOD score graphs created from the MAPMAKER output for Chr 17, using the combined data for both B and C rats. The X-
axes represent the recombination distance in centi-Morgans (cM). The horizontal dotted line represents the score required
for suggestive significance (1.9). a: plot for male rats and b: for female rats (solid lines). The combined male and female score
represented a locus that was between suggestive and full significance (dotted plot, LOD = 2.71). This locus also had a male spe-
cificity that on further analysis was shown to come from the male B rats (data not shown).






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a

4-


3-


2-

1-

J


0


C


IV







Cerebrospinal Fluid Research 2005, 2:2


Hence this may be a second gender-specific locus. The
male bias largely came from the B group with the male
LEW/Jms parent as described above.

In summary, the genotype analysis of B rats with a male
LEW/Jms parent and grandparent showed no chromo-
somal regions indicative of a locus for hydrocephalus,
with the possible exception of Chrl 7. On the other hand,
analysis using C rats with a female LEW/Jms parent and
grandparent showed a locus with significant linkage for
hydrocephalus on Chr2 that chiefly affected males. The
locus on rat Chr2 is situated at 217-218 Mb in band q41.
The region at and around the locus is homologous to
human chromosomes 1 and 4. Chromosome 17 linkage
was common to both rat groups. There was a region of
Chrl7 that was between the suggestive and fully signifi-
cant level for hydrocephalus when the data was com-
bined. In this case, the locus acted on both sexes but more
so with the male rats from the B group. The peak was sit-
uated close to D17Rat62 located at 83.5 Mb in band
17q12.3. This region is homologous to human 10p14 at
12.25 Mb.

Discussion
The LEW/Jms rat is a model for fetal-onset human hydro-
cephalus. In the human, ventriculomegaly, defined as
dilated lateral ventricle atria, can be detected by ultra-
sound examination from 20 weeks of gestation and some-
times earlier [18]. In some cases the dilatation remains
stable or resolves. In other cases there is progression to
hydrocephalus with increased head circumference and a
requirement for shunt treatment in the post-natal period.
Fetal hydrocephalus is frequently associated with a poor
neurodevelopmental outcome [18,19]. In many cases the
primary cause is uncertain, but stenosis of the cerebral
aqueduct is often a feature [20]. The LEW/Jms rat model
falls into this category, having fetal-onset progressive
hydrocephalus with an abnormal aqueduct [21,22]. In
many respects the phenotype is similar to hydrocephalus
in the H-Tx strain [23,24]. Both strains have severe fetal-
onset disease associated with aqueduct stenosis and dys-
plasia of the subcommissural organ [14,21,22]. Hydro-
cephalus expression in H-Tx rats has been shown to be
polygenic and influenced by at least four loci on different
chromosomes [12] and by strong epigenetic effects [25].
However, neither gender nor cross-specific effects were
observed in H-Tx. A surprising observation reported in
this study was the increase in the frequency with parity
from 22.2% in first litters to 47.6% in third litters. This
appears to be a similar phenomenon to that observed in
the H-Tx rat, where it was found that the frequency of
hydrocephalus was lower in first litters than in subsequent
litters [25]. In H-Tx hydrocephalus, the increase in hydro-
cephalus frequency among the pups in utero was associ-
ated with concurrent suckling by the dam of a previous


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litter. In the case of LEW/Jms rats, there was a progressive
increase in frequency with parity but whether or not the
phenomenon was related to concurrent suckling was not
investigated. It does indicate, however, that there may be
epigenetic effects affecting the expression of hydrocepha-
lus in this strain as occurs in the H-Tx strain.

The results of this study suggest that there is a locus for
hydrocephalus on Chr2, as shown in male rats with a
female LEW/Jms parent. There is possibly a second locus
on Chrl7 that is associated with hydrocephalus in rats
with parents of either sex, although the males with a male
LEW/Jms parent made the largest contribution. This is the
first time that a genetic analysis has been attempted in the
LEW/Jms rat strain. It was reported previously that twice
as many male as females rats are affected with hydroceph-
alus [11]. Although DNA samples from the BC1 progeny
with a male LEW/Jms parent were tested with seven ChrX
markers, only one marker, DXRat83 at position 43.2 Mb
in band q21 on the X chromosome (Ensembl Rat Genome
Browser http://www.ensembl.org), showed a low level of
significance (P < 0.05) with the male rats. It therefore
seems unlikely that X chromosome linkage is involved
despite the fact that X-linked hydrocephalus is well char-
acterized in humans [4] and is due to mutations in the
gene coding for L1 neural cell adhesion molecule. A more
likely explanation for the specific sex effects is that gender
affects phenotypic expression in this strain. Gender-spe-
cific loci have been observed in the analysis of other quan-
titative traits in rodents [26-28]. One explanation for this
phenomenon is that genes on the autosomes, such as
those coding for sex hormones, may influence the expres-
sion of disease-related genes.

In a recent genetic analysis of inherited hydrocephalus in
the H-Tx rat, a region of Chrl7 was identified at 25 55
cM that was highly significant for hydrocephalus in this
strain. It was concluded that one or more loci exist in this
region [12]. The equivalent genetic map positions were
71.2 92.6 Mb. This large region is homologous to
human Chrlq43 and 10p 11.21-p13. The locus identified
here for LEW/Jms rats is located in the middle of this sec-
tion, close to D17Rat62 located at 83.5 Mb in band
17q12.3. This region is homologous to human 10pl4 at
12.25 Mb (Fig. 5). It is possible that the two strains share
a common susceptibility locus for hydrocephalus on
Chrl7. There are three possible candidate genes in this
region close to D17Rat 62. One is SPAG6 at human
10pl2.2 and 22.6 Mb. This gene codes for sperm-associ-
ated antigen isoform 1 which is the murine homologue of
a component of the central flagella apparatus in sperm
flagellae. Spag6 knockout mice are infertile and have
hydrocephalus [29]. These mice may have impaired cilia
function in the brain, a potential cause for hydrocephalus
[30,31]. Two other known genes located within 10 Mb of


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Rat Chrl7
Mb
55
D17Rat23


60+


D17Rat79


D17Rat98 I
65-



D17Arb5
70-
D17Wox13
D17Ratl51
D17Rat34 I


75+


D17Rat89


D17Rat4180-.

D17Rat42
D17Rat62
85--


90

D17Rat85
D17Ratl54

95-


Human Homology


Chr



7


--FZD8 (10p11.21, 35.9)

-MTR (1q43, 233.3)

CHRM3 (1q43, 236.4)
H-Tx Peak 1




10





LEW/Jms Peak


ITGA8 (10p13, 15.7)


1-1-- VIM (10p13, 17.4)


-- H-Tx Peak 2
SPAG6 (10p12.2, 22.8)


Figure 5
A schematic map for the hydrocephalus locus on rat Chr 17. The scale on the left is the rat genetic length in megabases (Mb).
Positions for the rat DNA markers (open squares) were identified from Rat Genome Browser, Ensemble web site http://
www.ensembl.org. To the right of the rat chromosome are the human chromosome homologues. The positions for two possi-
ble hydrocephalus loci found in a previous study on H-Tx rats [12], and the peak for LEW/Jms rats in this study are marked
(arrows). Possible candidate genes (black diamonds and arrows) are named and their human cytogenetic and Mb positions
given in parenthesis.


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Rat Chr2 Human Homology

Mb Chr


D2Rat34


145-


D2Ratl 52
165-


D2Rat46


185-


D2Rat52


205-


D2Rat241


225
I


D2Rat62


245
D2Rat263


13


3


4-


S CA14 (1q21.2, 147.0)

- NOTCH2 (1p12, 120.1)

NGFB (1p13.1, 115.5)



LEW/Jms Peak C group

CGT (4q26, 116.0)

P97582, ANK2 (4q25, 114.3)


Figure 6
A schematic map for the hydrocephalus locus on rat Chr2. The scale on the left is the rat genetic length in megabases (Mb).
Positions for the rat DNA markers (open squares) were identified from Rat Genome Browser, Ensemble web site http:/
www.ensembl.org. To the right of the rat chromosome are the human chromosome homologues. The peak LOD score for
LEW/Jms rats is marked (arrows). Possible candidate genes (black diamonds and arrows) are named and their human cytoge-
netic and Mb positions given in parenthesis.


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Cerebrospinal Fluid Research 2005, 2:2


the locus are ITGA8 or integrin alpha 8 at human 10p13
and located at 15.6 Mb, and VIM coding for vimentin at
human 10pl3 and located at 17.3 Mb. Both of these genes
are important for brain development [32,33] but have no
known association with hydrocephalus. Three more can-
didate genes are located on this chromosome close to the
largest linkage peak for the H-Tx strain but further from
the LEW/Jms peak [12]. These are FDZ8, frizzled 8 precur-
sor which codes for a Wnt receptor, MTR, methyltetrahy-
drofolate-homocysteine methyltransferase or vitamin B2-
dependant methionine synthase, and the gene for the ace-
tylcholine muscarinic type 3 receptor, CHRM3.

The locus identified by MAPMAKER on Chr2 was located
close to marker D2Rat241 at 217-218 Mb in band q41.
This region is homologous with human Chrl and with a
section of human Chr4 (Fig 6). It is also homologous with
mouse Chr3 and includes a region that is particularly rich
in genes that are transcribed in the nervous system [34].
The regions at and around the locus were examined for
possible candidate genes using Ensemble Genome
Browser to identify rat genes and homologous human or
mouse genes that are expressed in brain and might have
an association with hydrocephalus. One such gene is
P97582 (rat 224.2 Mb) or ANK2 (human Chr4, 114.3
Mb), which codes for brain ankyrin or ankyrinB. Ankyrins
are spectrin-binding proteins on cell membranes that
associate with L1 CAM, and with several ion channels.
AnkyrinB deficient mice have a similar phenotype to L1
deficient mice with features that include dilated cerebral
ventricles [35]. Close by at 222.9 Mb is CGT or 2 hydroxy-
acyl sphingosine 1-B-galactosyltransferase, a gene found
in oligodendrocytes and involved in myelination [36].
Another attractive candidate is NGFB, or beta nerve
growth factor precursor at position 197.2 Mb. NGF and
other neurotrophins and their receptors are upregulated
in brain damage including that caused by hydrocephalus
[37]. Furthermore, hydrocephalic H-Tx rats have altera-
tions in brain NGF concentrations [38] and children with
hydrocephalus have elevated NGF in the CSF [39].
NOTCH2, or notch homologue protein 2 precursor, is at
192.8 Mb and close to NGFB. Notch proteins are trans-
membrane receptors involved in cell fate determination
in the CNS and Notch2 is important for roof plate devel-
opment [40]. Notch2 affects Wnt-1 expression, and in
mouse the Wnt sw/sw mutant has defective SCO develop-
ment and hydrocephalus [41]. Also in the same region of
Chr2 is CA14 or membrane-associated carbonic anhy-
drase XIV precursor at 190.65 Mb. This isoform of car-
bonic anhydrase is expressed in choroid plexus in
addition to neuronal cells but its function is not clear [42]
although another isoform CAII plays an important role in
CSF secretion at the choroid plexus [43].


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The identification of possible candidate genes is extremely
speculative because of the low resolution obtained from
QTL linkage analysis and the fact that the chromosomal
regions identified contain many hundreds of genes. In
contrast to genetic diseases with Mendelian inheritance,
QTL mapping for complex traits has not, in most cases,
led to the identification of abnormal genes [44]. However,
additional strategies are available such as expression pro-
filing in disease states, DNA sequencing for polymor-
phisms in candidate genes and transgenic technology all
of which can lead to gene identification. In conclusion,
the genetic basis for hydrocephalus expression in LEW/
Jms rats is associated with one or possibly two genetic loci
and in addition, the phenotypic expression is strongly
influenced by gender.

Competing interests
The authors) declare that they have no competing
interests.

Authors' contributions
HCJ conceived of the study, was responsible for its design
and coordination and writing the manuscript. DAM and
BJC participated in rat breeding. CFT, DAM, MY and BJC
all participated in phenotype and genotype analysis. CFT
was responsible for the male/female analysis, and assisted
in figure, table and manuscript preparation.

Acknowledgements
This research was funded by the Maren Foundation and NIH NS40359. We
are grateful for L. Morel for initial consultations. The technical assistance of
E. Joy Akins, Baligh Yehia and Gin Fu Chen is gratefully acknowledged.

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