Sequence analysis of 21 genes located in the Kartagener syndrome linkage region on chromosome 15q

Abstract

Primary ciliary dyskinesia (PCD) is a rare genetic disorder, which shows extensive genetic heterogeneity and is mostly inherited in an autosomal recessive fashion. There are four genes with a proven pathogenetic role in PCD. DNAH5 and DNAI1 are involved in 28 and 10% of PCD cases, respectively, while two other genes, DNAH11 and TXNDC3, have been identified as causal in one PCD family each. We have previously identified a 3.5 cM (2.82 Mb) region on chromosome 15q linked to Kartagener syndrome (KS), a subtype of PCD characterized by the randomization of body organ positioning. We have now refined the KS candidate region to a 1.8 Mb segment containing 18 known genes. The coding regions of these genes and three neighboring genes were subjected to sequence analysis in seven KS probands, and we were able to identify 60 single nucleotide sequence variants, 35 of which resided in mRNA coding sequences. However, none of the variations alone could explain the occurrence of the disease in these patients.

Full text

ARTICLE
Sequence analysis of 21 genes located in the
Kartagener syndrome linkage region on chromosome
15q
Maciej Geremek
1,2
, Frederieke Schoenmaker
1
, Ewa Zietkiewicz
2
, Andrzej Pogorzelski
3
,
Scott Diehl
4
, Cisca Wijmenga*
,1,5,7
and Michal Witt
2,6,7
1
Complex Genetics Group, Department of Biomedical Genetics, University Medical Center Utrecht, Utrecht, the
Netherlands;
2
Department of Molecular and Clinical Genetics, Institute of Human Genetics, Poznan, Poland;
3
Institute
of Tuberculosis and Lung Diseases, Rabka, Poland;
4
New Jersey Dental School, University of Medicine and Dentistry
New Jersey, Newark, NJ, USA;
5
Department of Genetics, University Medical Center Groningen, University of Groningen,
Groningen, the Netherlands;
6
International Institute of Molecular and Cell Biology, Warsaw, Poland
Primary ciliary dyskinesia (PCD) is a rare genetic disorder, which shows extensive genetic heterogeneity
and is mostly inherited in an autosomal recessive fashion. There are four genes with a proven pathogenetic
role in PCD. DNAH5 and DNAI1 are involved in 28 and 10% of PCD cases, respectively, while two other
genes, DNAH11 and TXNDC3, have been identified as causal in one PCD family each. We have previously
identified a 3.5 cM (2.82 Mb) region on chromosome 15q linked to Kartagener syndrome (KS), a subtype of
PCD characterized by the randomization of body organ positioning. We have now refined the KS
candidate region to a 1.8 Mb segment containing 18 known genes. The coding regions of these genes and
three neighboring genes were subjected to sequence analysis in seven KS probands, and we were able to
identify 60 single nucleotide sequence variants, 35 of which resided in mRNA coding sequences. However,
none of the variations alone could explain the occurrence of the disease in these patients.
European Journal of Human Genetics (2008) 16, 688– 695; doi:10.1038/ejhg.2008.5; published online 13 February 2008
Keywords: Kartagener syndrome; PCD; gene mapping; sequencing; mutation screening
Introduction
Primary ciliary dyskinesia (PCD, MIM no. 242650) is a rare
genetic disorder caused – as far as is known – by mutations
in genes encoding proteins important for ciliary beating.
1
The major clinical consequences fall into three categories:
(1) in the respiratory tract, immotility or dyskinetic beating
of cilia in epithelial cells impairs the mucociliary clearance,
leading to recurrent infections, sinusitis and bronchiectasis;
(2) in the urogenital tract, infertility occurs in some
patients because of the dysmotility of sperm tails; (3)
mirror reversal of body organ positioning (situs inversus) is
seen in approximately half of PCD patients. It is believed to
occur at random and to be caused by the immotility of
primary cilia of cells on the ventral surface of the
embryonic node. The presence of situs inversus in an
affected family defines a PCD subtype, known as
Kartagener syndrome (KS, MIM no. 244400).
For diagnostic purposes, cilia in bronchial scrapings can
be visualized under a light microscope, whereas the clinical
diagnosis of PCD is routinely verified by electron micro-
scopy analysis of respiratory cilia ultrastructure. About 20
different ultrastructural defects have been described
Received 8 June 2007; revised 11 December 2007; accepted 6 January
2008; published online 13 February 2008
*Correspondence: Professor C Wijmenga, Department of Genetics,
University Medical Center Groningen, University of Groningen, PO Box
30001, RB Groningen 9700, the Netherlands.
Tel: þ31 50 3617 100; Fax: þ31 50 3617 230;
E-mail: c.wijmenga@umcutrecht.nl
7
These authors have contributed equally to this work.
European Journal of Human Genetics (2008) 16, 688 – 695
&
2008 Nature Publishing Group All rights reserved 1018-4813/08
$30.00
www.nature.com/ejhg

in PCD patients, with lesions of outer and/or inner dynein
arms being the most frequent defects of the internal
anatomy of cilia.
2,3
Recently, it has been shown that
immunofluorescence staining of ciliated epithelium with
antibodies targeting DNAH5 can detect outer dynein arms
defects and therefore aid diagnosis of PCD.
4
Inheritance of PCD is autosomal recessive in most cases,
although pedigrees with an X-linked mode of inheritance
have also been described.
5,6
To date, mutations in four
genes have been found as causative for PCD, exclusively in
patients with outer dynein arm defects. DNAI1, coding
for intermediate dynein chain 1, was selected for mutation
screening based on homology with the Chlamydomonas
reinhardtii gene, which causes a slow-swimming phenotype
when mutated,
7,8
whereas DNAH5, coding for heavy
dynein chain 5, was identified by homozygosity mapping
in a large family and subsequent sequence analysis.
9
DNAH5 and DNAI1 are responsible for 28 and 10% of
PCD cases, respectively,
10,11
clearly indicating that other
genes are involved in PCD/KS etiology. DNAH11,
12
coding
for dynein heavy chain, and TXNDC3,
13
coding for a
thioredoxin family member, were also found to be
responsible for PCD, but so far each has been found in
only one family.
Analysis of the ciliary proteome in unicellular alga
C. reinhardtii led to the identification of a number of proteins
potentially involved in cilia formation and maintenance:
360 proteins with high and 293 with moderate confi-
dence.
14
Besides the known components of ciliary ultra-
structure, there were also 90 signal transduction proteins,
and many membrane proteins and metabolic enzymes.
Given that our knowledge of ciliary genes comes mostly
from studies of simple organisms and that the functional
spectrum of the possible candidate genes is very wide, it is
difficult to predict which of these should be considered
as candidate genes for PCD.
On the basis of genetic linkage studies, extensive genetic
heterogeneity of PCD is postulated. A total-genome scan
performed in 31 multiplex families did not reveal any
predominant locus, rather it showed several peaks with
suggestive and indicative LOD scores.
15
Another genome
scan, performed in five families of Arabic origin and with
reported consanguinity, revealed linkage with chromo-
some 19q13.3 in only three of the families, thus
confirming locus heterogeneity.
16
Two additional loci, on
16p12 and 15q, were indicated by studies on genetically
isolated, but heterogeneous, populations from the Faroe
Islands and the Israeli Druze.
17
We earlier performed genome-wide linkage analysis in 52
families with KS and reported a region on chromosome
15q24– 25, between D15S973 and D15S1037, linked to
KS.
18
After obtaining a significant LOD score of 4.34 with
D15S154 (using a strictly defined disease model), we
defined a region of 3.5 cM (2.82 Mb) as containing the
candidate gene.
18
In the present study, we have performed
further fine-mapping of the region of interest and screened
all 18 genes in the linked region and three neighboring
genes for mutations in seven KS patients from the families
that contributed most to the linkage results.
18
Materials and methods
Biological material
DNA samples from the existing PCD collection
18
came
from 31 Caucasian families (25 of Polish and 6 of Slovak
origin) classified as KS families; none was from a genetically
isolated population. Each family had at least one
member diagnosed with PCD and exhibiting situs inversus,
but with no other major anomalies or dysmorphologies
present. The group studied consisted of 38 affected
individuals and 99 unaffected family members. The
primary complaints in these patients were derived from
the respiratory tract and the clinical picture included
symptoms of sinusitis, nasal polyps, bronchiectasis, recur-
rent infections of the upper respiratory tract. All cases were
confirmed by a low concentration of nitric oxide (NO)
measured in exhaled air.
19
NO was measured from the
nasal cavity by a chemiluminescence analyzer with a
threshold value of 200 ppb for diagnosing PCD. In 60%
of the families, the clinical diagnosis was confirmed by
transmission electron microscopy analysis of bronchial
cilia ultrastructure; the analysis revealed defects of outer or
outer and inner dynein arms; a representative EM picture is
shown in Supplementary Figure 1. Direct microscopy of
bronchial scrapings was used to confirm the clinical
diagnosis of PCD in the patients that did not have electron
microscopy imaging of cilia. KS inheritance in these
pedigrees was in agreement with general assumptions for
an autosomal recessive disease model. KS in these families
has previously been shown to be linked with the region
between D15S973 and D15S1037, assuming full pene-
trance, 0.0001 phenocopies and the disease allele
frequency of 0.005. The genome-wide significant LOD
score of 4.34 was obtained using a cohort of 52 families.
18
Pair-wise LOD score analyses were performed using the
FASTLINK program, and multipoint LOD scores for markers
on chromosome 15 were calculated using GENEHUNTER.
Multipoint LOD scores allowing for locus heterogeneity
were calculated using Simwalk2. The 95% confidence
interval localized the KS locus to a 3.5 cM region between
D15S973 and D15S1037.
Recombination mapping using microsatellite markers
To refine the candidate gene region between D15S973
and D15S1037, seven markers (D15S1027–380 kb–
D15S524–33 kb–AFMA085AWB9–580 kb–D15S989–219 kb–
D15S1005–58 kb–D15S969–695 kb–D15S551), were geno-
typed in all 38 affected individuals and 99 unaffected
family members. Amplification products, obtained using
50end fluorescent-dye-labeled primers, were pooled and
Sequencing of 21 genes in 15q KS locus
M Geremek et al
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European Journal of Human Genetics

analyzed on an automated ABI Prism 3730 DNA sequencer
(Applied Biosystems, Foster City, CA, USA). Genotypes
were scored using GENMAPPER (v3.5NT) software. Inheri-
tance was checked using PEDCHECK
20
and haplotypes in
the 31 pedigrees were constructed from microsatellite data
using SIMWALK2.
21
Analysis of candidate genes
Sequence analysis was performed in seven affected indivi-
duals from the families with the highest LOD score
for marker D15S1005.
18
Sequences of 135 exons and
surrounding intron sequences of the 20 genes located in
the region were obtained using Ensembl genome browser
version 42 (Figure 1; Table 1). AL109678, not present in
Ensembl, was also sequenced as it is an mRNA coding
sequence annotated in the UCSC genome browser. At least
200 bp upstream of the transcription start site were also
analyzed and the 30UTR was sequenced in all genes except
for TMC3,KIAA1199,ARNT2 and TMED3. The Primer3
program was used for designing PCR primers yielding
products of 400 – 500 bp;
22
primers were in the intronic
sequences flanking the exons to be analyzed. PCR reactions
were performed using standard protocols (data available
upon request). Amplification products were purified,
quantified on a 2% agarose gel, and diluted for direct
sequencing on an automated ABI Prism 3730 DNA
sequencer using BigDye Terminator Sequencing Standards
(Applied Biosystems). Sequences were assembled using
ContigExpress (Vector NTI suite v8.0, Informax Inc.).
Sequencing reactions were performed first with the
forward primer, but if the quality of the sequencing
electropherogram was unsatisfactory, or if single-nucleo-
tide polymorphisms (SNPs) were found, they were repeated
with the reverse primer. Each of the newly identified
variants was validated through an independent PCR and
sequencing reaction. The sequenced fragments were only
included in the results if data were obtained for at least six
of the seven patients. Seven reactions could not be
amplified despite at least three attempts with two different
sets of primers (nucleotides in mRNA sequence: BCL2A1,
602– 710; FAM108C1, 1 – 343; C15ORF37, 421 –640;
TMED3, 260– 299; ENSG00000180725, 457 – 618; FAH,
683– 783, 991 – 1037). Reactions failed to use control as
well as patients’ DNA, indicating PCR problems related to
the local sequence composition of those genomic frag-
ments. All variations found in the protein-coding sequence
and not previously described in public databases were
sequenced in a control population group consisting of 48
unrelated, healthy individuals of Polish origin.
Results
Seven microsatellite markers located in the 3.5 cM region
reported to be linked to KS were genotyped in 31 families
that had positive LOD scores for chromosome 15q in our
genome linkage scan
18
(Figure 1). Haplotype analysis was
concordant with linkage to chromosome 15q24 – 25 (data
not shown). A recombination was mapped in family
numbers 114 and 126 (Figure 2), 1 Mb more telomerically
from the previous location defined by marker D15S937,
thus narrowing the minimal gene-containing region
down to 1.8 Mb, between markers AFMA085AWB9 and
D15S1037.
The analysis of haplotype sharing between nonrelated KS
patients was inconclusive. The most promising block,
encompassing three markers (D15S524, AFMA085WB9
and D15S989) and spanning 0.6 Mb, was present on five
nonrelated, affected chromosomes (families 103, 117, 136,
142 and 147) and on one untransmitted parental chromo-
some (data not shown).
Seven families with the highest positive LOD scores were
selected for sequence analysis of all the genes located in
TMC3
LOC390616
TMED3
K1024
MTHFS
Q9HBF5
C15ORF3
BCL2A1
ZFAND6
FAH
ENSG00000196548
ENSG00000205296 Q8N817 MESDC2
AL109678
ARNT2
FAM108C1
KIAA1199
MESDC1
C15ORF26
ENSG00000180725
D15S524
AFMA085WB9 D15S989 D15S969
D15S1005
D15S1037
D15S551
100kb
Figure 1 The location of 21 genes in the linkage region on chromosome 15q24-25. The minimal KS gene-containing region is indicated by an
arrow.
Sequencing of 21 genes in 15q KS locus
M Geremek et al
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European Journal of Human Genetics

the 1.8 Mb region (Figure 2). These families contributed
70% of the LOD score for marker D15S1005 on chromo-
some 15q,
18
and it was interesting that the DNAI1
mutation analysis was negative in all seven families. The
DNAH5 contribution was excluded in four families (114,
117, 136 and 147; Figure 2), based on the analysis of SNPs
located within the gene (discordant genotypes in the
affected sibs), while in the three remaining families, the
analyzed SNPs in DNAH5 were uninformative (unpub-
lished data). As the microsatellite data confirmed linkage of
the disease to 15q24– 25 in all seven families, only one
affected individual, representative of each pedigree, was
subjected to DNA sequencing. Supplementary Table 2
summarizes the clinical findings of the seven families
included for sequencing.
We analyzed the 18 coding sequences located between
markers AFMA085WB9 and D15S1037 (Table 1). We also
sequenced three genes located in the vicinity of the
region, which could be functionally related to cilia:
MESDC2, which is important in embryonic development;
C15ORF26, which is expressed in lung and testis and
displays homology to a flagellar protein identified in
Chlamydomonas;
14
and TMC3, a membrane protein with
partial homology to nephrocystine, as indicated by Blast
search of the NCBI nr database (Figure 1). Except for
C15ORF26, none of the genes have been reported to be a
part of cilium.
In total, we analyzed 83 kb of genomic sequence in seven
individuals (35 kb of coding sequences and 48 kb of
intronic/flanking DNA). We found 60 SNPs, 45 of which
were already present in public SNP databases. Twelve SNPs,
including the four previously unknown ones, were located
in the protein coding regions. None of the identified SNPs
in the coding regions resulted in a stop codon change or
Table 1 Genes included in the Kartagener syndrome sequencing study
Gene symbol Gene name GO description No. of exons
Transcript
length (bp)
LOC390616 Unknown Unknown 1 1608
AL109678 Unknown Unknown 1 2540
TMED3 Transmembrane emp24 domain-containing
protein 3 precursor (membrane protein p24B)
Protein transport; membrane;
membrane; protein carrier activity
3 1420
K1024 UPF0258 protein KIAA1024 Membrane; integral to membrane 4 6732
MTHFS 5-formyltetrahydrofolate cyclo-ligase (EC 6.3.3.2)
(5,10-methenyl- tetrahydrofolate synthetase)
(methenyl-THF synthetase) (MTHFS)
Magnesium ion binding; folic acid
binding; ligase activity; metabolism
3 857
ENS-
G00000205296
Unknown Unknown 2 153
Q9HBF5 Cervical cancer suppressor-1 Unknown 2 240
C15ORF37 C15orf37 protein Unknown 1 2082
BCL2A1 Bcl-2-related protein A1 (protein BFL-1)
(hemopoietic-specific early response protein)
(protein GRS)
Regulation of apoptosis 3 624
ENS-
G00000180725
Unknown Unknown 2 698
ZFAND6 Zinc-finger A20 domain-containing protein 3
(associated with PRK1 protein)
DNA binding; zinc ion binding;
signal transduction; protein
binding
7 1680
ENS-
G00000196548
Unknown Unknown 3 123
FAH Fumarylacetoacetase (EC 3.7.1.2)
(fumarylacetoacetate hydrolase) (beta-diketonase)
(FAA)
Fumarylacetoacetase activity;
magnesium ion binding; regulation
of transcription, DNA-dependent;
metabolism
15 1471
ARNT2 Aryl hydrocarbon receptor nuclear translocator 2
(ARNT protein 2)
Transcription factor activity; signal
transducer activity; aryl
hydrocarbon receptor nuclear
translocator activity; response to
hypoxia; embryonic development
19 6550
Q8N817 CDNA FLJ40133 fis, clone TESTI2012231 Unknown 4 675
FAM108C1 Unknown Unknown 3 2326
KIAA1199 KIAA1199 Sensory perception of sound 29 6602
MESDC2 Mesoderm development candidate 2 (NY-REN-61
antigen)
Mesoderm development 5 2938
MESDC1 Mesoderm development candidate 1 Unknown 1 2373
C15ORF26 Uncharacterized protein C15orf26 Unknown 7 1574
TMC3 Transmembrane channel-like protein 3 Integral to membrane 19 2443
Sequencing of 21 genes in 15q KS locus
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European Journal of Human Genetics

Figure 2 Pedigrees of the KS families included in the sequencing. The results of the pair-wise LOD score for marker D15S1005 are indicated below
each family. The recombination (marked by a red arrow) defining the minimal KS gene-containing region can be observed in pedigrees 114 and 126.
Individuals included in the sequencing are indicated by black arrows. Red dots indicate individuals exhibiting situs inversus. The numbers beside
marker names indicate the intermarker distances in kilobase pairs. Missing data are denoted by zeros. The genotypes of the parents in pedigrees 117
and 126 were deduced from the genotypes of the siblings.
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Table 2 List of all sequence variations found in 7 Kartagener syndrome patients
Gene Variation Amino acid Exon\intron
ID in SNP
database
Minor allele
frequency in NCBI
genome
database 35
Minor allele
frequency in 48
control individuals
of Polish origin
Number of patient
chromosomes with
the minor allele
LOC39016 c.735C4T P245P Exon 1 rs8038778 T ¼0.367 7/14
TMED c.659G4A A172A Exon 3 rs906439 A ¼0.0 0/14
KIAA1024 c.847A4C N258H
a
Exon 2 FC¼0.06 2/14
c.918C4A A281A Exon 2 F1/14
c.2569A4G I832V
b
Exon 3 rs2297773 G ¼0.208 9/14
c*1799A4CF30UTR rs17266017 C ¼0.375 2/14
c.*3771_3772insATG F30UTR FIns ¼0.06 2/14
BCL2A1 c.238G4A C19Y
c
Exon 1 rs1138357 A ¼0.146 3/14
c.299T4G N39K
d
Exon 1 rs1138358 G ¼0.146 3/14
MTHFS g.411A4GFUpstream rs2865825 F1/14
g.421C4GFUpstream rs2903105 F3/14
g.483delG FUpstream rs35401897 F2/14
C15ORF37 c.903C4A P166Q
f
Exon 1 rs3803540 A ¼0.217 1/14
c.*46G4TF30UTR rs17214656 T ¼0.167 2/14
c.*124A4CF30UTR rs36070199 F2/14
c.*206A4GF30UTR rs12442408 G ¼0.250 1/14
c.*413A4TF30UTR rs2733101 T ¼0.283 2/14
AWP1 c.29590T4CFIntron 2 rs1916048 C ¼0.217 5/12
C574+11C4GFIntron 5 rs1522636 G ¼0.261 9/14
c.46660A4TFIntron 4 rs12915043 F1/14
c.506A4G Q65Q Exon 4 FG¼0.0 1/14
FAH c.915173delC FIntron 10 rs3835063 F6/14
c.1133C4T S352S Exon 13 rs1801374 T ¼0.058 2/14
c.103835A4CFIntron 12 rs2043691 C ¼0.08 1/14
c.1038223C4TFIntron 12 rs2043692 T ¼0.15 1/14
c.*37A4CF30UTR rs1049181 F2/14
c.*38C4TF30UTR FF 2/14
c.*39C4TF30UTR rs3210172 F2/14
c.*40C4TF30UTR FF 2/14
c.*41C4GF30UTR FF 2/14
c.*50delC F30UTR FF 2/14
c. *93T4CF30UTR rs1049194 F2/14
ARNT2 c.312+15C4TFIntron 3 rs2278708 T ¼0.058 1/14
c.36128insC FIntron 3 rs5814026 F3/14
C574+36C4GFIntron 4 rs8033507 G ¼0.017 1/14
c.89274C4TFIntron 7 rs11072922 T ¼0.175 2/14
c.1255+21C4TFIntron 11 rs3924894 T ¼0.405 8/14
c.1255+22T4CFIntron 11 rs3924893 C ¼0.4 6/14
c.1279T4A W399R
e
Exon 12 - A ¼0.0 1/14
FAM108C1 c.*692T4CF30UTR rs1046417 C ¼0.130 2/14
c.*1115C4AF30UTR rs11072940 A ¼0.104 2/14
KIAA1199 c.255T4AF50UTR rs2273886 A ¼0.295 4/14
c.1029+26C4GFIntron 7 FF 7/14
c.1346+240C4TFIntron 9 FF 1/14
c.1479+83T4AFIntron 10 FF 1/14
c.2057+57A4GFIntron 13 rs2271164 G ¼0.417 5/12
c.2142T4C C28C Exon 14 rs2271160 C ¼0.261 3/14
c.2462A4CFIntron 16 rs2271162 C ¼0.167 3/14
MESDC2 c.*3G4TF30UTR rs8039607 T ¼0.121 1/14
c.*53G4TF30UTR rs8039384 T ¼0.142 1/14
c.136920_136918del-
CTT
FIntron 4 FDel ¼0.08 1/14
c.*1416C4TF30UTR FT¼0.069 1/14
c.*2153T4CF30UTR rs10519305 C ¼0.017 1/14
C15ORF26 G496C4GFUpstream rs2683255 F1/14
c.505105insA FIntron 3 FF 3/14
c.50587C4G Intron 3 FF 3/14
c.66450insA Intron 5 rs11395672 F7/14
TMC3 c.13T4CF50UTR FC¼0.08 1/14
c.580+55T4C Intron 6 rs12903128 C ¼0.345 1/14
c.82366A4G Intron 7 rs7167034 A ¼0.153 7/14
Nucleotide variations are presented according to the HUGO nomenclature. Translation start site was used for each gene as the +1 nucleotide. Putative
effect on protein:
a
Small, polar4polar, charged, positive, aromatic;
b
Aliphatic4aliphatic, small;
c
Tiny4aromatic;
d
Small4positive;
e
Aromatic, polar4polar, positive;
f
Small4polar.
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a frame shift; six were amino-acid substitutions and seven
were synonymous codon changes. Twenty-one SNPs,
including seven novel ones, were located in the untrans-
lated regions. Four SNPs were located in regions upstream
of the transcription start site. Twenty-seven variations were
located in noncoding regions, six of which were previously
unknown. None of the SNPs found were closer than 10 bp
to a splicing site. Variations in the protein-coding
sequence, previously not described in SNP databases, were
sequenced in 48 individuals of Polish origin to estimate
population frequencies. Except for two SNPs (W399R in
ARNT2 and A506G in AWP1), all the variations were found
in the healthy controls. A complete list of the SNPs found is
given in Table 2 and the distribution of all SNPs in the
patients can be found in Supplementary Table 1.
Discussion
We have performed a follow-up study to our previous
finding
18
of linkage to chromosome 15q24 – 25 in families
with KS. Fine-mapping of the 3.5 cM (2.82 Mb) linked
region in 31 KS families allowed us to narrow down the
minimal candidate gene region to 1.8 Mb. Analysis of
haplotypes composed of microsatellite markers revealed
that there was no extensive haplotype sharing among the
affected individuals, arguing against the presence of only
one or a few frequent mutations occurring on a common
haplotype background. On the other hand, the absence of
haplotype sharing does not exclude the possibility of the
presence of a causative gene or genes in the linked region.
The linkage analysis is independent of a founder effect.
There are examples in literature of genes involved in rare
recessive diseases, but not showing evidence of haplotype
sharing. This happens because most of the patients are
compound heterozygous for unique mutations in the given
gene.
23
In searching for the KS gene in the 15q24-25 region, we
found 37 SNPs in the mRNA-coding sequences and
promoters of 14 genes, including 6 nonsynonymous codon
changes. No evidently pathogenic mutations, such as the
introduction of stop codons or frame-shift mutations, were
detected.
Given the rare occurrence of the disease (approximately
1:40 000), the expected cumulative frequency of all the
disease variant(s) in the general population should be
below 1%. We also sequenced fragments harboring variants
in mRNA-coding sequence in 96 control chromosomes
(Table 2). One nonsynonymous amino-acid change
(W399R in ARNT2), and one silent codon change (A506G
in AWP1) were not seen in the controls, suggesting that
their frequencies might be as low as 1%. However, each of
these two variants (in two different genes) was found in
heterozygous form in only a single patient, with no
other possibly pathogenic complementary allele; these
are therefore unlikely to represent the disease-causing
mutations. None of the remaining SNPs were particularly
rare and their occurrence in patients did not significantly
deviate from population frequencies (Table 2).
In conclusion, we did not find any variants that directly
fulfilled the criteria of a pathogenic mutation for KS in
any of the 21 genes located in the minimal gene-contain-
ing region, as indicated by linkage analysis (while
assuming the rare occurrence of the disease and the
recessive mode of inheritance). The analyzed region may,
however, contain pathogenic variants that we failed to
find. We did not sequence introns that could contain
regulatory elements or harbor gain-of-splicing site muta-
tions. It is also possible that some of the genes have
alternative exons not annotated in the current databases
and thus not analyzed in our study, and we cannot exclude
the possibility that some of the genes harbor heterozygous
deletion(s) of one or more exons. We did not detect any
extended homozygous regions, in neither the micro-
satellite data nor in the SNP data, but the relatively low
density of heterozygous SNPs could have left some regions
noninformative with respect to their purported hemi-
zygosity (see Supplementary Table 1).
As the pedigree structures in the cohort we studied were
simple, we cannot exclude the possibility that some of the
families are false positives, although none of the families
was crucial for the significant LOD score. Hence, despite
the statistical significance of the LOD score value, it cannot
entirely be excluded that the 15q locus is a false positive
locus. The current location of the linkage interval is based
on two families (114 and 126; Figure 2) that contribute to
the left recombination and on one family
18
that contri-
butes to the right recombination. This last family contains
only one affected individual and, therefore, contributed
very little to the overall LOD score. Under less stringent
disease models, the linkage peak would still be significant
with a 95% confidence interval matching the minimal
gene-containing region. However, the linkage curve would
be positive over a larger region with more than 200 genes.
Hence, we cannot exclude that the KS gene is located closer
to the 15q telomere. Given that this larger region does not
contain obvious candidate genes, more families will be
needed to establish the exact KS region.
Acknowledgements
We thank all the Polish and Slovak PCD families who participated in
this study for their invaluable cooperation, and J Pawlik (Rabka,
Poland) and A Kapellerova (Bratislava, Slovakia) for providing study
material and clinical data. We gratefully acknowledge the help of Ewa
Rutkiewicz in collecting biological material, of Jackie Senior in editing
the manuscript and of Behrooz Alizadeh in interpreting the statistical
data. This research was partly financed by Grant PBZ-KBN-122/
P05-1 from the Polish Ministry of Science and Higher Education. MG
is supported by the International PhD Program of the University of
Utrecht.
Sequencing of 21 genes in 15q KS locus
M Geremek et al
694
European Journal of Human Genetics

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Supplementary Information accompanies the paper on European Journal of Human Genetics website (http://www.nature.com/ejhg)
Sequencing of 21 genes in 15q KS locus
M Geremek et al
695
European Journal of Human Genetics