Department of Clinical and Experimental Medicine
Division of Clinical Genetics
Final Thesis
PCR Optimisation and Sequencing of L1CAM for the Verification
of a Mutation in a Family with L1 Syndrome
Malin Eriksson
LiU-IKE-EX—09/08
Department of Clinical and Experimental Medicine Linköpings universitet
Department of Clinical and Experimental Medicine
Division of Clinical Genetics
Final Thesis
PCR Optimisation and Sequencing of L1CAM for the Verification
of a Mutation in a Family with L1 Syndrome
Malin Eriksson
LiU-IKE-EX—09/08
Supervisors: Cecilia Gunnarsson
Clinical Genetics, Linköping University Hospital
Katarina Ellnebo Svedlund
Clinical Genetics, Linköping University Hospital Examiner: Olle Stål
Department of Clinical and Experimental Medicine, Linköping Univeristy
Linköping 090119
Department of Clinical and Experimental Medicine Linköpings universitet
Defence date
Publishing date (Electronic version)
Department and Division
ISBN: ISRN: Title of series Language
English
Other (specify below) ________________ Report category Licentiate thesis Degree thesis Thesis, C-level Thesis, D-level Other (specify below)
__________________
Series number/ISSN
URL, Electronic version
Title
Author(s)
Abstract
Keywords
2009-01-19 Department of Clinical and
Experimental Medicine
LiU-IKE-EX—09/08
http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-16954
PCR Optimisation and Sequencing of L1CAM for the Verification of a Mutation in a Family with L1 Syndrome
Malin Eriksson
L1 syndrome is an X-linked recessive disorder, characterised by congenital hydrocephalus, adducted thumbs, spastic paraplegia, agenesis of the corpus callosum and mental retardation. The disease is caused by mutations in the L1CAM gene, encoding a protein predominantly expressed in the nervous system. This protein has been implicated in a variety of processes including neuronal migration, neurite outgrowth and fasciculation and myelination.
L1 syndrome was suspected at genetic counselling of a family with a boy suffering from
congenital hydrocephalus and mental retardation. Complete sequencing of L1CAM, performed by an external laboratory, revealed a novel mutation in the family, including a boy, affected with L1
syndrome, his sister, his mother and his maternal grandmother.
To verify this mutation and to be able to detect mutations in the L1CAM gene locally in the future, a method for mutational analysis of this gene was set up using PCR optimisation and cycle sequencing.
Sequencing of L1CAM was then performed on DNA samples from the four family members and the mutation was verified. A point mutation (c.3458-1G>C) in the 5’ splice site of exon 26 was
detected in all of them. This new, not previously described, mutation is supposed to cause a deletion of the 26th exon and a frameshift in the part of the protein encoded by exons 27 and 28. This means that almost the entire cytoplasmic domain of the protein would have a loss of function, explaining the symptoms affecting the boy.
Abstract
L1 syndrome is an X-linked recessive disorder, characterised by congenital hydrocephalus, adducted thumbs, spastic paraplegia, agenesis of the corpus callosum and mental retardation. The disease is caused by mutations in the L1CAM gene, encoding a protein predominantly expressed in the nervous system. This protein has been implicated in a variety of processes including neuronal migration, neurite outgrowth and fasciculation, myelination, and long-term memory formation.
L1 syndrome was suspected at genetic counselling of a family with a boy suffering from congenital hydrocephalus and mental retardation. Complete sequencing of L1CAM, performed by an external laboratory, revealed a novel mutation in the family, including a boy, affected with L1 syndrome, his sister, his mother and his maternal grandmother.
To verify this mutation and to be able to detect mutations in the L1CAM gene locally in the future, a method for mutational analysis of this gene was set up using PCR optimisation and cycle sequencing.
Sequencing of L1CAM was then performed on DNA samples from the four family members and the mutation was verified. A point mutation (c.3458-1G>C) in the 5’ splice site of exon 26 was detected in all of them. This new, not previously described, mutation is supposed to cause a deletion of the 26th exon and a frameshift in the part of the protein encoded by exons 27 and 28. This means that almost the entire cytoplasmic domain of the protein would have a loss of function, explaining the symptoms affecting the boy.
Table of Contents
1 INTRODUCTION ... 1
1.1 AIM ... 3
2 MUTATIONAL ANALYSIS – THEORETICAL BACKGROUND ... 5
2.1 DNAEXTRACTION ... 5
2.2 AMPLIFICATION OF THE ENTIRE GENOMIC DNA ... 6
2.3 PURIFICATION OF AMPLIFIED GENOMIC DNA ... 6
2.4 POLYMERASE CHAIN REACTION,PCR ... 7
2.5 CAPILLARY ELECTROPHORESIS ... 7
2.6 PCRREACTION CLEANUP ... 8
2.7 CYCLE SEQUENCING REACTION ... 9
2.8 SEQUENCING REACTION CLEANUP ... 11
2.9 CYCLE SEQUENCING ... 11
2.10 ANALYSIS ... 12
3 MATERIALS AND METHODS ... 13
3.1 CLINICAL DESCRIPTION ... 13 3.2 SAMPLES ... 14 3.3 PRIMER SELECTION ... 14 3.4 DNAEXTRACTION ... 16 3.5 DNAAMPLIFICATION ... 17 3.6 DNAPURIFICATION ... 17 3.7 PCRREACTION ... 18 3.8 CAPILLARY ELECTROPHORESIS ... 21 3.9 PCRREACTION CLEANUP ... 21
3.10 CYCLE SEQUENCING REACTION ... 22
3.11 SEQUENCING REACTION CLEANUP ... 23
3.12 CYCLE SEQUENCING ... 23 3.13 ANALYSIS ... 23 4 RESULTS ... 25 4.1 PCR ... 25 4.2 SEQUENCING ... 25 5 DISCUSSION ... 29 5.1 CONCLUSION ... 30 6 ACKNOWLEDGMENTS ... 33 7 REFERENCES ... 35
1 Introduction
The L1 cell adhesion molecule (L1CAM) gene is located in the Xq28 region near the telomere of the long arm of the X chromosome (Djabali et al., 1990). L1CAM consists of 29 exons of which 28 include coding regions (Kallunki et al., 1997) encoding transmembrane glycoprotein belonging to the immunoglobulin superfamily (IgSF) (Moos et al., 1988; Hlavin and Lemmon, 1991).
The L1 protein is made up of an extracellular part consisting of six immunoglobulin-like (Ig-like) domains and five fibronectin type III-like (FnIII) domains, a single-pass transmembrane domain, and a short cytoplasmic C-terminal tail (Figure 1) (Moos et al., 1988; Hlavin and Lemmon, 1991).
Figure 1. Schematic representation of the L1CAM protein consisting of six immunoglobulin-like domains, five
fibronectin type III-like domains, one transmembrane domain and one cytoplasmic domain (L1CAM Mutation Web Page).
L1 is expressed on neurons, both in the central nervous system and in the peripheral nervous system (Kenwrick et al., 2000). On differentiated neurons L1 is found at regions of contact between neighbouring axons and on the growth cones, which is the highly motile, membranous structure at the leading tip of axons that are responsible for sensing extracellular growth and guidance cues (Figure 2).
Non-neural expression of an alternatively spliced L1 lacking both exon 2 and exon 27 has been described in the male urogenital tract (Kujat et al., 1995), in the intestinal crypt cells (Thor et al., 1987), in a subclass of leukocytes (Kowitz et al., 1992) and in kidney tubule epithelia (Debiec et al., 1998). Expression of L1 has also been described in cells of tumoural origin (Fogel et al., 2003a; Fogel et al., 2003b; Deichmann et al., 2003; Thies et al., 2002; Miyahara et al., 2001).
L1 has been implicated in a variety of important processes including neuronal migration (Lindner et al., 1983), neurite outgrowth (Lagenaur and Lemmon, 1987) and fasciculation
(bundling) (Stallcup and Beasley, 1985), myelination (Wood et al., 1990), neural cell survival (Chen et al., 1999), synaptogenesis and growth cone morphology (Burden-Gulley et al., 1995). In addition, L1 is involved in the establishment of long-term potentiation (which is a use dependent increase in synaptic efficacy implicated in learning and memory) (Lüthl et al., 1994), long-term memory formation (Rose, 1995) and in regeneration of damaged nerve tissue (Martini and Schachner, 1988). The most common mode of action is probably homophilic L1-L1 binding between adjacent membranes of neurons (Kamiguchi et al., 1998a). However, a variety of heterophilic binding partners for L1 has also been suggested. These heterophilic binding partners include TAG-1/axonin-1 (Felsenfeld et al., 1994; Kuhn et al., 1991) and F3/F11 - two other Ig superfamily adhesion molecules (Morales et al., 1993), a
proteoglycan called phosphocan (Grumet et al., 1993), and an integrin called αvβ3
(Montgomery et al., 1996).
Figure 2. The structure of a neuron.
The function of L1 in tissues outside the nervous system, however, is unclear (Fransen et al., 1997).
An X-linked recessive disorder called L1 syndrome is caused by mutations in L1CAM (Kanemura et al., 2006). The major features include congenital hydrocephalus (accumulation of cerebrospinal fluid in the ventricles of the brain, causing progressive enlargement of the head) (Figure 3a), adducted thumbs (Figure 3b), spastic paraplegia (progressive weakness and spasticity in the muscles in the lower body), agenesis of the corpus callosum (a complete or partial absence of the corpus callosum) (Figure 3c) and mental retardation (Finckh et al., 2000). Since L1 syndrome is an X-linked recessive disorder, it predominantly affects males. Fewer than 5 % of females carrying an L1CAM mutation manifest clinical features (Kaepernick et al., 1994). These features are usually minor symptoms of the clinical spectrum, although severe hydrocephalus has been reported in a female carrier.
Figure 3. Symptoms of L1 syndrome. Hydrocephalus (a) (Health Library). Adducted thumb (b) (Liebau et al.,
2007). Agenesis of the corpus callosum. The corpus callosum connects the left and right cerebral hemispheres (c) (The New York Times).
According to the latest (October, 2005) listing of L1CAM mutations on the L1CAM Mutation Web Page (L1CAM Mutation Web Page), 178 different mutations have been identified in 206 unrelated families in which one individual has been diagnosed with L1 syndrome.
Currently, DNA sequencing is a standard technique for detecting gene mutations and it is suggested that direct sequencing is a good way to examine all the L1CAM exons rapidly and to obtain reproducible results (Kanemura et al., 2006).
Recently, Clinical Genetics at Linköping University Hospital came in contact with a family in which three members were having symptoms of L1 syndrome. Complete sequencing of
L1CAM was performed by an external laboratory and a novel mutation was detected in the
family, including a boy with expected L1 syndrome, his sister, his mother and his maternal grandmother.
1.1 Aim
To verify this mutation and to be able to detect mutations in the L1CAM gene locally in the future a method for mutational analysis of this gene had to be set up. The aim of this final thesis is to optimise the PCR and the sequencing of L1CAM for this purpose and to discuss the possible effects of this mutation.
2
Mutational Analysis – Theoretical Background
2.1 DNA
Extraction
The BioRobot EZ1 System (QIAGEN AB, Solna, Sweden) is used to automatically extract DNA from, for example, whole blood samples treated with EDTA (QIAGEN, 2008a). First the blood cells are lysed using a lysis buffer (Figure 4). Then DNA is isolated from the lysates by binding to the silica surface of magnetic particles. The magnetic particles are then separated from the lysates using a magnet. The DNA is washed and finally eluted in elution buffer.
2.2 Amplification of the Entire Genomic DNA
The illustra™ GenomiPhi™ V2 DNA Amplification Kit (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) is used to amplify the entire genomic DNA (GE Healthcare, 2006a). First, the DNA is denaturated and mixed with random hexamers that non-specifically bind to the DNA. Then, Phi29 DNA polymerase, additional random hexamers, nucleotides, salts and buffers are added and the amplification begins. The amplification procedure is shown in Figure 5.
Figure 5. Overview of the GenomiPhi V2 DNA Amplification Kit procedure (GE Healthcare, 2006a).
2.3 Purification of Amplified Genomic DNA
The illustra™ GFX™ PCR DNA and Gel Band Purification Kit (GE Healthcare Bio-Sciences AB) is used to purify the amplified genomic DNA (GE Healthcare, 2007). The procedure is supposed to denaturate proteins and to remove salts and other contaminants from the sample.
2.4 Polymerase Chain Reaction, PCR
Overlapping fragments covering the exons and splice sites of the L1CAM gene is amplified by the means of PCR. The amplification is performed in fragments because the entire gene cannot be sequenced in one reaction.
2.5 Capillary
Electrophoresis
The QIAxcel system (QIAGEN) uses capillary gel electrophoresis for size-based separation of nucleic acids (QIAGEN, 2008b). The separation is performed in a capillary of a precast gel cartridge. There are twelve capillaries in one cartridge. The samples are automatically loaded into an individual capillary and voltage is applied. The negatively charged nucleic acid molecules migrate through the capillary to the positively charged terminus (Figure 6). Low-molecular-weight molecules migrate faster than high-Low-molecular-weight molecules. As the molecules migrate through the capillary, dye molecules attach to the DNA and fluoresce when subjected to light-emitting diodes (LEDs). The DNA molecules then pass a detector which detects and measures the fluorescent signal. A photomultiplier converts the fluorescent signal into electronic data, which are then transferred to the computer workstation for further processing using the BioCalculator software. After processing, the data are displayed as an electropherogram and as a gel image.
Capillary electrophoresis is used to verify that fragments have been amplified and that they are of the right size. Each PCR product should result in one band on the gel image (or one peak in the electropherogram). If there are more bands the by-products may interfere in the sequencing.
Figure 6. Sample separation process using the QIAxcel system (QIAGEN, 2008b). Nucleic acid molecules are
size separated by applying an electrical current to a gel-filled capillary. A photomultiplier detector in the QIAxcel instrument detects the dyed nucleic acid molecules as they migrate towards the positively charged terminus of the cpillary. The data are converted to an electropherogram and a gel image by the BioCalculator software.
2.6 PCR Reaction Cleanup
The PCR products are cleaned using ExoSAP-IT® (GE Healthcare Bio-Sciences AB). The
procedure involves two hydrolytic enzymes, Exonuclease I and Shrip Alkaline Phosphatase, that are used together to remove unused PCR primers and unincorporated nucleotides from PCR products without any sample loss (Figure 7) (GE Healthcare, 2006b). Exonuclease I degrades single-stranded primers and any single-stranded DNA produced in the PCR and Shrip Alkaline Phosphatase dephosphorylates the remaining dNTPs from the PCR mixture. Without this PCR cleanup procedure the excess primers and nucleotides can present problems in dye terminator chemistry and cause noisy sequencing data later on (Applied Biosystems, 2006).
Figure 7. Schematic diagram of the ExoSAP-IT method (GE Healthcare, 2006b). The PCR results in PCR
products as well as in unused PCR primers and unincorporated dNTPs. ExoSAP-IT degrades the excess PCR primers and dephosphorylates the excess dNTPs to prevent noisy analysis data.
2.7 Cycle Sequencing Reaction
Today, a protocol referred to as “cycle sequencing” is the method of choice for determining DNA sequence (Lodge et al., 2007). Cycle sequencing exploits the polymerase chain reaction, fluorescent labeling and capillary electrophoresis. The cycle sequencing reaction is set up in two tubes, one for the forward reaction and one for the reverse reaction. The double-stranded template DNA is mixed with primer (reverse or forward), deoxynucleoside triphosphates (dNTPs), thermostable DNA polymerase and the four dideoxynucleoside triphosphates (ddNTPs). The ddNTPs differs from dNTPs in the way that no nucleotides can attach to them. Therefore, these modified dideoxynucleosides are often called terminators. Each ddNTP is labelled with a different fluorescent dye, so when the dye terminator is used by DNA polymerase it will lead to both chain termination and labelling of the DNA fragment, the label indicating which base was last incorporated in the growing chain. The sample is then
amplified using a thermal cycler. In this amplification, the forward primer and the reverse primer are used separately. This way, there will be a linear amplification of one strand of the DNA. The sequence is then received through the use of capillary electrophoresis. A schematic representation of a cycle sequencing reaction is shown in Figure 8.
Figure 8. The cycle sequencing reaction. The template DNA is mixed with DNA polymerase, primers, dNTPs
and the four ddNTPs with varying fluorescence. When the DNA polymerase incorporates a ddNTP into the chain, there will be both a chain termination and a labelling that indicates which base was last incorporated. This amplification will result in numerous fragments of varying lengths and fluorescence. The sequence is then received using capillary electrophoresis.
2.8 Sequencing Reaction Cleanup
Before the sequencing reaction products are analysed using the capillary electrophoresis, the
products are purified using the BigDye® XTerminator™ Purification Kit (Applied
Biosystems, Stockholm, Sweden). The kit is designed to separate cycle-sequencing reaction components, such as salt ions, unincorporated dye terminators, and dNTPs, from dye-labelled extension products to prevent their co-injection (Applied Biosystems, 2007). Without this step the components may cause noisy sequencing data.
2.9 Cycle
Sequencing
Next, the reaction is analysed by capillary electrophoresis. This is not the same capillary electrophoresis as the one used for analysing the PCR products, but the principles are the same. A typical capillary is 50 µm in diameter and 50 cm in length (Lodge et al., 2007). Prior to loading the sample the capillary is filled with a polymer, the precise formula of which will vary depending on the supplier. The polymer will act as the molecular filter during electrophoresis. The sample is taken up by one end of the capillary and a voltage is applied, the DNA sample will migrate towards the positive electrode or anode. Smaller DNA fragments will migrate through the capillary faster than larger fragments. The capillary is capable of resolving DNA fragments that vary in size by just one base pair; a requirement for the sequencing to be successful. At the end of the capillary is a laser beam that will hit each fluorescently labelled DNA fragment as it comes out of the capillary. The laser will cause the fluorescent dyes to fluoresce, this fluorescence is detected by a charged couple device (CCD) camera. The data from the camera is then processed by a computer to generate an electropherogram with the fluorescence plotted against time. Each peak on the electropherogram will represent a DNA fragment of a specific length. The lengths of the fragments along with the different fluorescence of the four terminators make up the sequence of the DNA sample. The principle of the capillary electrophoresis is shown in Figure 9.
Figure 9. Capillary electrophoresis. The technique is used to convert the fluorescent fragments of different
lengths to a DNA sequence.
2.10 Analysis
Finally, the received data is analysed using the SeqScape Software (Applied Biosystems), which compares the obtained sequences against a reference sequence and displays the differences and possible mutations.
3
Materials and Methods
3.1 Clinical
Description
The patient was the second child of healthy nonconsanguineous Swedish parents. On the
mother’s side, there was a family history of males with hydrocephalus and adducted thumbs, see pedigree (Figure 10). In the patient, hydrocephalus was first detected by fetal ultrasound at 36 weeks gestation. The patient was delivered at 38 weeks gestation by Caesarian and mild global hypotonia (a condition of abnormally low muscle tone) and bilateral adducted thumbs
were noted soon after birth, as was the hydrocephalus. Karyotype revealed a normal 46, XY
male karyotype. A ventriculoperitoneal shunt (a surgery performed to draw off the fluid from the ventricles of the brain into the abdomen and thereby relieving the pressure inside the skull,
caused by the hydrocephalus) was placed at 2 days of age. The family history revealed the
two maternal uncles with hydrocephalus, spastic paraplegia and adducted thumbs. The boy’s
mother was diagnosed with clear cell renal cell carcinoma (ccRCC) at the age of 46, and died two years later. The mother was seeking for genetic counselling when the boy was 20 years old and his older sister was 23 years old. Informed consent was obtained from the family
members involved in the study. Due to the clinical signs of hydrocephalus and adducted
thumbs among males in this family, L1 syndrome was suspected. Blood samples from the boy (patient 3), the sister (patient 4), the mother (patient 1) and the mother’s mother (patient 2) were collected for complete sequencing of L1CAM. This sequencing was performed by Centogene (Rostock, Germany) and the results showed that all four family members had a mutation in the gene.
Figure 10. Pedigree of the family. The arrow points out the proband, in this case the mother. The numbered
family members are the ones who were analysed for the mutation.
3.2 Samples
First, pooled DNA samples extracted from blood from three individuals with no known mutation in the L1CAM gene was used. These samples were used for the optimisation of the PCR to not consume the entire amount of patient DNA. Then, two individual DNA samples from healthy patients were used to confirm the PCR conditions and to optimise the sequencing. To verify the mutation already found in the family members (Patient 1-4), DNA samples from patients 1, 2 and 4, and a blood sample from patient 3 out of which DNA was extracted were used.
In addition, two DNA samples from a patient diagnosed with breast cancer were used to evaluate whether the PCR would be successful using DNA from paraffin embedded and fresh frozen tissue.
3.3 Primer
Selection
The PCR primers were chosen using the NCBI Probe database, according to a protocol from Applied Biosystems (Applied Biosystems, 2006). They were chosen to cover the entire coding sequence and the splice sites of the gene. The PCR fragments produced by the primer mixtures cover the gene according to Figure 11.
Figure 11. The gene with its exons (black) and introns (white) and the locations of the PCR fragments produced
by the primer mixtures. The exons are sorted 1-28 from left to right. The fragments (grey) are sorted 1-33 from right to left.
M13 tail sequences were added to the primer sequences to simplify the sequencing later on. The sequencing is simplified in the way that M13 primers can be used in the sequencing reaction and this way the same sequencing primers can be used for all the PCR products. The primer sequences are shown in Table 1.
Table 1. The primer mixtures consisting of forward and reverse primers with the M13 tail sequences in lower
case letters.
Primermix
number Forward primer Reverse primer
Product size
1 tgtaaaacgacggccagtCCTGGCAGGTCGCTCCTAGC caggaaacagctatgaccGGCCTCCGTCACTACCTGGC 558
2 tgtaaaacgacggccagtCACTAGTGG
CGTAAAGGGAAGGACA
caggaaacagctatgaccCCGAAA
GGGTCTCCGAAATGC 599
3 tgtaaaacgacggccagtGAGTGCGATGCTGGGAGTGG caggaaacagctatgaccCCCTGCCGTGGCCCTAGAAT 590
4 tgtaaaacgacggccagtGGACCGGGTGGTAGGAAGGG caggaaacagctatgaccTGCCCAAAGATGACAGCTCCA 533
5 tgtaaaacgacggccagtGGACCGGG TGGTAGGAAGGG caggaaacagctatgaccAACAAA TGGAAGGCAGGCGG 464 6 tgtaaaacgacggccagtACTGAACAT CCACGCTGCCC caggaaacagctatgaccGGCCAA CAGCAAGGTCTCCC 562
7 tgtaaaacgacggccagtCGTGTTGGCCTCTCCCTGAA caggaaacagctatgaccACAGGCAAGTGGTGGCTGGA 505
8 tgtaaaacgacggccagtCACCCGGCAGCACAGAGAAG caggaaacagctatgaccCCCTTCTGCAAGGCCTCCTG 429
9 tgtaaaacgacggccagtCCACCATGC
GCCTGTCATCT
caggaaacagctatgaccCGGCACC
AAATGGCTGTGAA 598
10 tgtaaaacgacggccagtAGGCCTTGCAGAAGGGTGGA caggaaacagctatgaccGCCACTACTGCCCAGGCTCC 441
11 tgtaaaacgacggccagtGGGCATCTGCGGAAAGGGTAT caggaaacagctatgaccTGCCACACTCTCCTCGTTCCC 599
12 tgtaaaacgacggccagtGGGCATCTGCGGAAAGGGTA caggaaacagctatgaccCTCAGCCACAGCGGGTGAAA 542
13 tgtaaaacgacggccagtGGTGCCGG
AACATCCTCTCC
caggaaacagctatgaccTTCGGAC
ACACAACCTGACCG 594
14 tgtaaaacgacggccagtTGCAGGTCCCACTGCGTGTA caggaaacagctatgaccGCAACTGTCCTTCAACCTTCGG 573
16 tgtaaaacgacggccagtGAGCACGT AGCCGGTGAGCA
caggaaacagctatgaccAAAGCG
ACAGGATGGTGAGGG 557
17 tgtaaaacgacggccagtTGACACTGGTGGTGTTGGCG caggaaacagctatgaccCGAATTCGTCTTCTCTGTGTGTAGGG 505
18 tgtaaaacgacggccagtTGAAAGAAGCAGCATTGGCTGA caggaaacagctatgaccGGCCATGACCTGGGTGTCTG 578
19 tgtaaaacgacggccagtCACCAGCAT
TCTTTGGCCCG
caggaaacagctatgaccGTCTGGG
CAAGGTTCCAGGG 590
20 tgtaaaacgacggccagtTCTGGCACCAAGGGAGTCCA caggaaacagctatgaccGCTTGAGCTCAGTGCCACCC 582
21 tgtaaaacgacggccagtGTTCCCTGGAACCTTGCCCA caggaaacagctatgaccGGCACAGCTCTTGGTGGTGG 537
22 tgtaaaacgacggccagtGCTGGCAGAAGTGACGGTGG caggaaacagctatgaccCCTCTTGGCCTCGTCCTTGC 494
23 tgtaaaacgacggccagtGCCATCTGG
GCTCTTCTCCC
caggaaacagctatgaccAGGCTG
GACGAGGATGGGAC 578
24 tgtaaaacgacggccagtCATGTGGCAAGGGTTGCCTG caggaaacagctatgaccGAGAGACACAGCCTGGCGGG 569
25 tgtaaaacgacggccagtGCGGTCCGCTCAGTCACAGT caggaaacagctatgaccGAGGGTGAGCAGGGCCTCAG 511
26 tgtaaaacgacggccagtGCATTGAGC
TGCGTTGAGGC
caggaaacagctatgaccGGGTGAT
TGGCCTTGTCCTTTC 595
27 tgtaaaacgacggccagtAGTGAGTTCTCGGCCAGGCA caggaaacagctatgaccCCAGGCGTCGAGAGCAGAGA 555
28 tgtaaaacgacggccagtTCTCTGCTCTCGACGCCTGG caggaaacagctatgaccTGTCAGCCCGTCTGTCCCTT 573
29 tgtaaaacgacggccagtCGGCACCCACTGCTGTTCAT caggaaacagctatgaccACCCTTCCTGGCCTCCTGGT 372
30 tgtaaaacgacggccagtGTGGAGGG
CTGGCAGGACAC
caggaaacagctatgaccCAAGTGT
GAGGCCAGTGGCAA 595
31 tgtaaaacgacggccagtGACAGGTGCAAGCAGCCAGG caggaaacagctatgaccTGCCATTCTCTCTGGTCCCTTC 596
32 tgtaaaacgacggccagtGGTGCTCAGGGAGAGCCGAG caggaaacagctatgaccAGGCATCCTGAATGGGTGGG 537
33 tgtaaaacgacggccagtAGCATGGA
GGAGGCCCAAGA
caggaaacagctatgaccGCCCGG
GCTTACCCAGATGT 573
3.4 DNA
Extraction
Genomic DNA was extracted from blood samples, treated with EDTA, using BioRobot EZ1 (QIAGEN AB) and EZ1 DNA Blood 350 µl Kit (QIAGEN AB). DNA was extracted from 350 µl blood and eluated into 200 µl elution buffer.
The DNA concentrations were measured at 260 nm, using NanoDrop ND-1000 (Saveen Werner AB, Malmö, Sweden).
3.5 DNA
Amplification
Since there was a limited amount of DNA from the family members, an attempt to amplify the
entire genomic DNA was made. The DNA was amplified using the illustra™ GenomiPhi™
V2 DNA Amplification Kit (GE Healthcare Bio-Sciences AB), according to the manufacturer. 9 µl Sample Buffer was mixed with 1 µl of 10 ng DNA. The DNA was then denaturated by heating the samples to 95ºC for 3 minutes using PCT-100™ Programmable Thermal Controller (SDS Promega, Nacka, Sweden) and then cooled to 4ºC on ice. 9 µl Reaction Buffer was then, on ice, mixed with 1 µl Enzyme Mix. This mix was then transferred to the cooled samples and the samples were incubated at 30ºC for 1.5 hours (using the same thermal controller). The samples were then heated to 65ºC for 10 min to inactivate the Phi29 DNA polymerase enzyme. The amplified DNA did not result in PCR products with all PCR primers and therefore a purification of the amplified samples were performed before performing the PCR anew.
3.6 DNA
Purification
The amplified DNA was purified using the illustra™ GFX™ PCR DNA and Gel Band Purification Kit (GE Healthcare Bio-Sciences AB) according to the manufacturer. 500 µl Capture buffer type 2 was mixed with 60 µl amplified DNA and loaded on a GFX MicroSpin column placed into a Collection tube. The assembled column and Collection tube was then
spun at 13400 x g for 30 sec using the microcentrifuge Eppendorf MiniSpin® (VWR
International AB, Stockholm, Sweden) and the flow through was discarded. 500 µl Wash buffer type 1 was then added to the column, which was then spun at 13400 x g for 30 sec again. The Collection tube was then discarded and the column was transfered to a 1.5 ml microcentrifuge tube. 50 µl Elution buffer type 6 was added to the centre of the membrane in the column and incubated at room temperature for 1 minute. The assembled column and microcentrifuge tube was then spun at 13400 x g for 1 minute to recover the purified DNA. However, the amplified DNA did not result in PCR products for all primer mixtures after this purification procedure either. Because the DNA amplification and purification procedures were not included in the aim of this final thesis, no further effort was made to make them successful. Instead, the PCR and the sequencing were performed directly on DNA obtained from the DNA extraction.
3.7 PCR
Reaction
Amplification of L1CAM was performed by the means of PCR according to the Variant SEQr protocol (Applied Biosystems). PCR was performed using 0.25 µl genomic DNA (36.6 ng/µl)
as the template in a 10 µl reaction mixture, including 5 µl 2X AmpliTaq Gold® PCR Master
Mix (containing AmpliTaq Gold® DNA Polymerase, dNTPs and Gold Buffer) (Applied Biosystems) 1.6 µl 50 % UltraPure™ Glycerol (Invitrogen AB, Lidingö, Sweden) 1 µl 0.6 µM forward primer (Invitrogen AB) and 1 µl 0.6 µM reverse primer (Invitrogen AB). Water of PCR quality was added to adjust the reaction volume. The PCR amplification program consisted of one cycle of heat activation at 96ºC for 5 min, followed by 40 cycles of denaturation 94ºC 30 sec, annealing 60ºC 45 sec and extension 72ºC 45 sec ending with a final extension of 72ºC for 10 min and then held at 4ºC using the thermo cycler system, Eppendorf Mastercycler® EP (VWR International AB).
This reaction conditions resulted in products using primer mixtures 3, 5-10, 15-28 and 30-32. Optimisation had to be performed on the remaining primer mixtures.
First an alteration in primer and DNA concentration was evaluated. The primer concentration was set to 6 µM with 0.25 µl genomic DNA (36.6 ng/µl) and 1.25 µl genomic DNA (36.6 ng/µl) respectively. The water volume was adjusted to have a total volume of 10 µl.
After this step, only primer mixtures 1, 2, 29 and 33 were without products. They were then evaluated with a primer concentration of 9 µM and the original amount of genomic DNA (0.25 µl DNA 36.6 ng/µl).
With still no products using these four primer mixtures, the PCR Optimization Kit (Roche Diagnostics Scandinavia AB, Bromma, Sweden) was used according to the manufacturer’s protocol. 0.25 µl genomic DNA (36.6 ng/µl) was mixed with 0.20 µl 10 mM dNTP (Roche Diagnostics Scandinavia AB), 0.05 µl Taq DNA polymerase (5 U/µl) (Roche Diagnostics Scandinavia AB), 1 µl 6 µM forward primer (Invitrogen AB) and 1 µl 6 µM reverse primer (Invitrogen AB) and 6.50 µl water of PCR quality. Then 1 µl of buffers A-L, each containing
100 mM Tris-HCl and 500 mM KCl but with varying MgCl2 concentrations and pH-values as
Table 2. The PCR optimisation buffers (Roche Diagnostics Scandinavia AB). pH MgCl2 concentrations [mM] 1.0 1.5 2.0 2.5 8.3 A B C D 8.6 E F G H 8.9 I J K L 9.2 M N O P
This approach resulted in products using primer mixture 2 with buffer J, primer mixture 29 with buffer E, and primer mixture 33 with buffer I.
Primer mixture 1 would still not result in a product and was evaluated some more. The primer mixture was mixed as above with buffer J using the varying annealing temperatures, 60.1ºC, 63.2ºC, 66.0ºC and 69.4ºC.
This approach was no success either. With still no product, primer mixture 1 was mixed with the same base components as before (Table 3) with the addition of buffer J and buffer N (separately) and with a variety of additives included in the PCR Optimization Kit (Table 4 and 5). The original annealing temperature of 60ºC was used.
Table 3. PCR components with volumes and concentrations.
Component Volume Concentration
dNTP 0.20 µl 10 mM
Taq DNA polymerase 0.05 µl 5 U/µl
Genomic DNA 0.25 µl 36.6 ng/µl
Forward primer 1 µl 6 µM
Reverse primer 1 µl 6 µM
Optimisation buffer N, J 1 µl
Additive
Water, PCR Grade Filled up to a volume of 10 µl
Table 4. Additives 1.
Additive Volume Concentration
DMSO 1 µl 100 %
Gelatine 1 µl 1 %
H2O 1 µl
Table 5. Additives 2.
Additive Volume Concentration
DMSO 1 µl 100 % Glycerol 3 µl 50 % Gelatine 1 µl 1 % (NH4)2SO4 0.6 µl 500 mM MgCl2 1 µl 2.5 mM H2O 1 µl
Then, only buffer J was used with the varying additives shown in Table 6 and with the annealing temperatures 58.4ºC, 60.0ºC, 62.6ºC, 65.3ºC, 67.4ºC, 68.0ºC.
Table 6. Additives 3.
Additive Volume Concentration (original)
DMSO 1 µl 10 %
Glycerol 1 µl 50 %
Gelatine 1 µl 0.1 %
(NH4)2SO4 1 µl 50 mM
H2O 1 µl
After this, a number of different gelatine concentrations were tested according to Table 7. with buffer J and annealing temperatures 65ºC, 66ºC, 66.5ºC, 67ºC, 67.5ºC, 68ºC.
Table 7. Gelatine concentrations.
Volume Concentration 1 µl 0.01 % 0.5 µl 0.1 % 1 µl 0.1 % 0.5 µl 1 % 1 µl 1 % 1 µl 0 %
Then, yet another set of additives was tried, varying DMSO concentrations according to Table 8, and varying annealing temperatures 60ºC, 62ºC, 64ºC, 66ºC, 67ºC and 68ºC.
Table 8. DMSO concetrations.
Volume Concentration
1 µl 100 %
5 µl 10 %
1 µl 10 %
After considering the positions of the primer fragments it was decided to only go forward with primer mixtures 3-33. The first two primer mixtures produce fragments that are located after the last exon and splice site of the gene (Figure 11) and should not be important for receiving the DNA sequence.
The following PCR conditions were confirmed to be successful and were used on patient samples (family members 1-4):
For amplification with primer mixtures 3-28 and 30-32 PCR was performed using 0.25 µl genomic DNA (36.6 ng/µl) as the template in a 10 µl reaction mixture, containing 5 µl
5-10, 15-28 and 30-32, and 6 µM for primer mixtures 4, 11, 12, 13 and 14. Water of PCR quality was added to adjust the reaction volyme. The PCR amplification program consisted of one cycle of heat activation at 96ºC for 5 min, followed by 40 cycles of denaturation 94ºC 30 sec, annealing 60ºC 45 sec and extension 72ºC 45 sec ending with a final extension of 72ºC for 10 min and then held at 4ºC.
For amplification with primer mixtures 29 and 33 the PCR Optimization Kit was used. 0.25 µl genomic DNA (36.6 ng/µl) was mixed with 0.20 µl 10 mM dNTP, 0.05 µl Taq DNA polymerase (5 U/µl), 1 µl 6 µM forward primer and 1 µl 6 µM reverse primer, and 6.50 µl water of PCR quality. Then 1 µl of buffer E was added for amplification with primer mixture 29 and 1µl of buffer I was added for amplification with primer mixture 33. The PCR amplification program was the same as for the other primer mixtures described above.
3.8 Capillary
Electrophoresis
The PCR products were analysed using the capillary electrophoresis device, QIAxcel. The QX DNA Size Marker 50-800 bp (QIAGEN AB) and the QX Alignment Marker 15bp/3 kb (QIAGEN AB) were used for the analysis of the length of the PCR products.
3.9 PCR Reaction Cleanup
The PCR products were diluted depending on how intense the bands received from the capillary electrophoresis were. Bands like nr 1 in Figure 12 were diluted 1:5, bands like nr 2 were diluted 1:10 and bands like nr 3 were diluted 1:15. These dilutions were decided after some optimisation. Different dilutions were tested, 1:1, 1:5, 1:7.5, 1:10; 1:10, 1:15, 1:20; and 1:10, 1:15 respectively. If the samples are not diluted, the signals in the sequence analysis will be too intense and there will be no result.
1 2 3
Figure 12. Gel image of capillary electrophoresis results showing different intensities of the PCR products. The
bands of the sizes 15 and 3000 base pairs are alignment markers.
To purify the PCR products, 2 µl ExoSAP-IT® (GE Healthcare Bio-Sciences AB) was added
to 10 µl of diluted PCR product according to the VariantSEQr protocol from Applied Biosystems. The samples were then incubated at 37ºC for 30 min, heat inactivated at 80ºC for 15 min and then held at 4ºC using the same thermo cycler as before.
3.10 Cycle Sequencing Reaction
The purified PCR amplification products were then mixed into a sequencing reaction mixture using BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), according to the
manufacturer. The reaction mixture contained 1 µl BigDye® Terminator v3.1 Ready Reaction
Mix (containing dNTPs and dye terminators, enzyme, and sequencing buffer (2.5X)), 1.5 µl
BigDye® Terminator v1.1, v3.1 5X Sequencing Buffer, 1 µl 3.2 µM M13 forward primer
(Invitrogen AB) or 1 µl 3.2 µM M13 reverse primer (Invitrogen AB) and 4.5 µl deionised ultra filtrated water. 2 µl purified PCR product was then added to this mix. The mixture was then heat activated at 96ºC for 1 min, followed by 25 cycles of denaturation at 96ºC for 10 sec, annealing at 50ºC for 5 sec and extension at 60ºC for 4 min. The temperature was then held at 4ºC.
3.11 Sequencing Reaction Cleanup
The sequencing mix was then cleaned using BigDye® XTerminator™ Purification Kit (Applied Biosystems). 10 µl vortexed XTerminator™ Solution was mixed with 45 µl SAM™ Solution and then added to the sequencing mixture. Wide-bore pipette tips (tips with an orifice > 0.1 mm) were used for pipetting the Xterminator Solution. The mixture was then vortexed using the Eppendorf Thermomixer comfort (VWR International AB) at 22ºC and 1400 rpm for 1 h.
Then, the samples were centrifuged using Sigma 4K15 (LABEX, Helsingborg, Sweden) at 1000 rpm for 2 min.
3.12 Cycle Sequencing
10 µl of the supernatants were then transferred to a 96-well plate for sequence analysis using the 16-capillary electrophoresis DNA sequencer, ABI 3130xl Genetic Analyzer, (Applied Biosystems). The DNA sequencing was carried out in both directions for each template.
3.13 Analysis
The results were analysed using the SeqScape® Software v2.6 (Applied Biosystems)
4 Results
4.1 PCR
Products were obtained for all the primer mixtures (3-33) and they were of expected size, indicating that the products were the correct ones (Figure 13).
Figure 13. Gel image of capillary electroporesis showing the PCR products of the different primer mixtures
numbered 3-33.
PCR of DNA from paraffin embedded breast cancer tissue gave almost no products. This is probably due to a destroying of the DNA when it is obtained or when it is embedded. The fragments that are obtained are probably too short for the PCR to function correctly. The PCR may be successful if primers that produce shorter templates are used.
PCR of DNA from fresh frozen breast cancer tissue was as successful as the ones performed on DNA from blood samples. In other words, all PCR products were obtained using this material.
4.2 Sequencing
The L1CAM gene was successfully sequenced and the mutation already found in the family was verified. The mutation consists of a transversion of one nucleotide; a C instead of a G in the in the 5’splice site of exon 26 in the position closest to the exon (c.3458-1G>C). This mutation has not been reported previously. The mutation was found in all tested family members (Figure 14). In the three females, the mutation was found in heterozygosity and in the male in homozygosity since males only have one copy of the X chromosome.
Figure 14. The sequencing results of the region around the mutation from the SeqScape Software for all family
members. The numbering is the same as in Figure 10. The reference sequence is shown in lower case letters at the top. The S indicates that there are both a C (blue) and a G (black). The green colouring of the marked letters indicate that the nucleotides differ from the reference. Family members 1, 2 and 4 (females) have both a C and a G in this position and are all carriers of the mutation. Member 3, the male, only has one copy, a C, thus has the disease.
This mutation should cause a deletion of the entire 26th exon. Exon 26 encodes a part of the L1 protein that is located in the cytoplasmic domain (Figure 15).
Figure 15. The coding region with the exons of L1CAM numbered from 1 to 28 and the protein domains. (Data
from GenBank, M77640, Hlavin and Lemmon, 1991.)
Exon 26 consists of a number of nucleotides that are not dividable by three; therefore a deletion of this exon should also cause a frameshift leading to an incorrect amino acid sequence in the cytoplasmatic domain of the protein encoded by exons 27 and 28. This frameshift also causes an early stop codon, leading to the loss of two additional amino acids.
This means that the mutation, the one transversion, should cause a loss of function of almost the entire cytoplasmic domain.
5 Discussion
The gene was successfully sequenced and a point mutation in the 5’splice site of exon 26 was found in all four family members, verifying the mutation already found in the family. The mutation probably causes a loss of function of almost the entire cytoplasmic domain.
Since the boy and his uncles suffered from congenital hydrocephalus, adducted thumbs, spastic paraplegia and mental retardation, all symptoms of the L1 syndrome, this cytoplasmic domain of L1 must be very important for the protein to function in this family.
The cytoplasmic domain of L1 is multifunctional (Fransen et al., 1998). It provides an anchor to the cytoskeleton via ankyrin (Hortsch, 1996), a linker protein of the spectrin-based cytoskeleton that underlies the plasma membrane (Davis and Bennett, 1994; Hortsch et al., 1998). The interaction between the L1CAM cytoplasmic domain and ankyrin-B has been reported to be required for the initial protrusion of axons from the neuronal soma (Nishimura et al., 2003). Disturbance of these functions of the L1CAM cytoplasmic domain would cause the symptoms related to axon growth that occur in L1 syndrome.
The cytoplasmic domain contains serine phosphorylation sites (Hortsch, 1996). Phosphorylation may be a way for modulation of L1 signal transducting activity (Kunz et al., 1996; Schuch et al., 1989; Wong et al., 1996a; Wong et al., 1996b) and for the initiation of intracellular second messenger cascades (Schuch et al., 1989; Williams et al., 1994), which may be involved in L1 dependent neuronal movement.
The cytoplasmic domain of L1CAM also houses a tyrosine-based sorting motif that is required for the correct trafficking of L1 along axons to the growth cone (Kamiguchi et al., 1998b ) as well as for L1 endocytosis (Kamiguchi et al., 1998c). Clearly, mutations that delete this sorting motif will disrupt trafficking of L1 in differentiated neurons, only allowing transport of protein to the cell soma (Kenwrick et al., 2000).
In summary, mutations in the cytoplasmic domain would be expected to disrupt cytoskeleton interactions, L1-mediated signalling and L1 trafficking in the nervous system.
It is, therefore, likely that the mutation in L1CAM found in the family is the cause of the symptoms that are affecting the boy and that were also affecting his two deceased uncles.
This conclusion is supported by other mutations affecting the cytoplasmic domain of the L1 protein that have been reported to cause the same symptoms (L1CAM Mutation Web Page).
The mother in the family developed ccRCC, which was considered unusual for her age. This raised the question of a possible association between this disease and the mutation in L1CAM as well.
Expression of L1 has been found in the renal epithelium and it is suggested that L1 is involved in multiple stages of renal epithelial morphogenesis and that mutations in L1CAM may lead to development of abnormalities affecting the kidney (Debiec et al., 1998).
It has also been indicated that mutations in L1CAM may be responsible for duplex kidneys in a boy with L1 syndrome, supporting that mutations in the gene may cause renal malformations (Liebau et al., 2007).
L1 expression has been detected in ccRCC, renal tumours originating from cells that do not express L1 in the normal kidney (Allory et al., 2005). In addition, a truncated form of L1 lacking a part of its cytoplasmic tail has been detected in these tumours. In ccRCC, the presence of L1 has also been shown to associate with a higher risk of metastasis.
Since the expression of L1 has been shown to correlate with ccRCC, it is possible that the mutation occurring in the family may cause the ccRCC of the mother in the family. The detection, in these tumours, of a form of L1 lacking a part of the cytoplasmic domain is also interesting, since the mutation found in the family should cause the loss of function of the cytoplasmic domain, which is basically the same as lacking the domain.
The observation of this family will continue and the girl carrying the heterozygous mutation will be examined every fifth year with renal ultrasonography. The girl will also be offered prenatal diagnosis in case of future pregnancy.
5.1 Conclusion
The L1CAM gene was successfully sequenced and a novel mutation (c.3458-1G>C) was found causing L1 syndrome in the boy and his two uncles. Although it seems like the mutation causes a deletion of exon 26 and thereby a frameshift in the part of the protein encoded by exons 27 and 28, it could be an idea to confirm this by studying the mRNA.
The mother in the family, carrying the mutation, was affected with ccRCC and it is possible that the mutation in L1CAM is involved in this disease as well. A possible correlation like this remains to be investigated. Sequencing may be performed on DNA samples extracted from fresh frozen tumour tissue from patients with ccRCC in whom no other oncogen has been found to cause the disease.
6 Acknowledgments
First, I would like to thank Cecilia Gunnarsson and Jon Jonasson for giving me the opportunity to write this final thesis and the staff at the Division of Clinical Genetics for their support.
I would especially like to thank my supervisor Cecilia Gunnarsson for helping me with the theoretical work and my supervisor Katarina Ellnebo Svedlund for helping me with the practical work.
Thanks to Sofie Eriksson for proofreading.
Thanks to my examiner Olle Stål and to my opponent Lisa Sjöberg for valuable comments on my report.
7 References
Allory, Y., Matsuoka, Y., Bazille, C., Christensen, E. I., Ronco, P. and Debiec, H., 2005. The L1 Cell Adhesion Molecule Is Induced in Renal Cancer Cells and Correlates with Metastasis in Clear Cell Carcinomas. Clin Cancer Res. 11:1190-1197.
Applied Biosystems, 2006. VariantSEQr and mitoSEQr Resequencing Systems Protocol. http://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/ cms_041392.pdf. 2009-01-13.
Applied Biosystems, 2007. Big Dye XTerminator Purification Kit Protocol. http://docs.appliedbiosystems.com/pebiodocs/04374408.pdf. 2009-01-13.
Burden-Gulley, S. M., Payne, R. and Lemmon, V., 1995. Growth Cones Are Actively Influenced by Substrate-Bound Adhesion Molecules. J Neurosci. 15:4370-4381.
Chen, S., Mantei, N., Dong, L. and Schachner, M., 1999. Prevention of Neuronal Cell Death by Neural Adhesion Molecules L1 and CHL1. J Neurobiol. 38:428-439.
Davis, J. Q. and Bennett, V., 1994. Ankyrin Binding Activity Shared by the
Neurofascin/L1/NrCAM Family of Nervous System Cell Adhesion Molecules. J Biol Chem. 269:27163-27166.
Debiec, H., Christensen, E. I. and Ronco, P. M., 1998. The Cell Adhesion Molecule L1 Is Developmentally Regulated in the Renal Epithelium and Is Involved in Kidney Branching Morphogenesis. J Cell Biol. 143:2067-2079.
Deichmann, M., Kurzen, H., Egner, U., Altevogt, P. and Hartschuh, W., 2003. Adhesion Molecules CD171 (L1CAM) and CD24 are Expressed by Primary Neuroendocrine Carcinomas of the Skin (Merkel Cell Carcinomas). J Cutan Pathol. 30:363-368.
Djabali, M., Mattei, M. G., Nguyen, C., Roux, D., Demengeot, J., Denizot, F., Moos, M., Schachner, M., Goridis, C. and Jordan, B. R., 1990. The Gene Encoding L1, a Neural Adhesion Molecule of the Immunoglobulin Family, Is Located on the X Chromosome in Mouse and Man. Genomics. 7:587-593.
Felsenfeld, D. P., Hynes, M. A., Skoler, K. M., Furley, A. J. and Jessell, T. M., 1994. TAG-1 Can Mediate Homophilic Binding, But Neurite Outgrowth on TAG-1 Requires an L1-Like Molecule and β1 Integrins. Neuron. 12:675-690.
Finckh, U., Schröder, J., Ressler, B., Veske, A. and Gal, A., 2000. Spectrum and Detection Rate of L1CAM Mutations in Isolated and Familial Cases with Clinically Suspected L1-Disease. Am J Med Genet. 92:40-46.
Fogel, M., Gutwein, P., Mechtersheimer, S., Riedle, S., Stoeck, A., Smirnov, A., Edler, L., Ben-Arie, A., Huszar, M. and Altevogt, P., 2003a. L1 Expression as a Predictor of Progression and Survival in Patients with Uterine and Ovarian Carcinomas. Lancet. 362:869-875.
Fogel, M., Mechtersheimer, S., Huszar, M., Smirnov, A., Abu-Dahi, A., Tilgen, W., Reichrath, J., Georg, T., Altevogt, P. and Gutwein, P., 2003b. L1 Adhesion Molecule (CD
171) in Development and Progression of Human Malignant Melanoma. Cancer Lett. 189:237-247.
Fransen, E., Van Camp, G., Vits, L. and Willems, P. J., 1997. L1-Associated Diseases: Clinical Genetics Divide, Molecular Genetics Unite. Hum Mol Genet. 6:1625-1632.
Fransen, E., Van Camp, G., D’Hooge, R. Vits, L. and Willems, P. J., 1998. Genotype-Phenotype Correlation in L1 Associated Diseases. J Med Genet. 35:399-404.
GE Healthcare, 2006a. illustra GenomiPhi V2 DNA Amplification Kit Product Web Protocol. http://www4.gelifesciences.com/aptrix/upp00919.nsf/Content/AC0AECC3796CD8EDC1257 154007B2E1B/$file/GPHI_V2_25660030_revB.pdf. 2009-01-13.
GE Healthcare, 2006b. ExoSAP-IT PCR Clean-up Kit Data File. http://www4.gelife sciences.com/applic/upp00738.nsf/vLookupDoc/341819971-O150/$file/18115014AB.pdf. 2009-01-13.
GE Healthcare, 2007. illustra GFX PCR DNA and Gel Band Purification Kit Product booklet. http://www1.gelifesciences.com/aptrix/upp00919.nsf/Content/EF527125F24048B9C12572BF 0081201D/$file/28903470PL_Rev_C_2007_WEB.pdf. 2009-01-13.
Health Library. http://healthlibrary.epnet.com/GetContent.aspx?token=2e7354b6-ae71-4dab-90df-c7026eb1c66f&chunkiid=11771. 2009-01-13.
Hlavin, M. L. and Lemmon, V., 1991. Molecular Structure and Functional Testing of Human L1CAM: An Interspecies Comparison. Genomics. 11:416-423.
Hortsch, M., 1996. The L1 Family of Neural Cell Adhesion Molecules: Old Proteins Performing New Tricks. Neruon. 17:587-593.
Hortsch, M., O’Shea, K. S., Zhao, G., Kim, F., Vallejo, Y. and Dubreuil, R. R., 1998. A Conserved Role for L1 as a Transmembrane Link Between Neuronal Adhesion and Membrane Cytoskeleton Assembly. Cell adhes commun. 5:61-73.
Kaepernick, L., Legius, E., Higgins, J. and Kapur, S., 1994. Clinical Aspects of the MASA Syndrome in a Large Family, Including Expressing Females. Clin Genet. 45:181-185.
Kallunki, P., Edelman, G. M. and Jones, F. S., 1997. Tissue-Specific Expression of the L1 Cell Adhesion Molecule Is Modulated by the Neural Restrictive Silencer Element. J Cell Biol. 138:1343-1354.
Kamiguchi, H., Hlavin, M. L. and Lemmon, V., 1998a. Role of L1 in Neural Development: What the Knockouts Tell Us. Mol Cell Neurosci. 12:48-55.
Kamiguchi, H. and Lemmon, V., 1998b. A Neuronal Form of the Cell Adhesion Molecule L1 Contains a Tyrosine-Based Signal Required for Sorting to the Axonal Growth Cone. J
Neurosci. 18:3749-3756.
Kamiguchi, H., Long, K. E., Pendergast, M., Schaefer, A. W., Rapoport, I., Kirchhausen, T. and Lemmon, V., 1998c. The Neural Cell Adhesion Molecule L1 Interacts with the AP-2 Adaptor and is Endocytosed via the Clathrin-Mediated Pathway. J Neurosci. 18:5311-5321.
Kanemura, Y., Okamoto, N., Sakamoto, H., Shofuda, T., Kamiguchi, H. and Yamasaki, M., 2006. Molecular Mechanisms and Neuroimaging Criteria for Severe L1 Syndrome with X-Linked Hydrocephalus. J Neurosurg. 105:403-412.
Kenwrick, S., Watkins, A. and De Angelis, E., 2000. Neural Cell Recognition Molecule L1: Relating Biological Complexity to Human Disease Mutations. Hum Mol Genet. 9:879-886. Kowitz, A., Kadmon, G., Eckert, M., Schirrmacher, V., Schachner, M. and Altevogt, P., 1992. Expression and Function of the Neural Cell Adhesion Molecule L1 in Mouse Leukocytes. Eur
J Immunol. 22:1199-1205.
Kuhn, T. B., Stoeckli, E. T., Condrau, M. A., Rathjen, F. G. and Sonderegger, P., 1991. Neurite Outgrowth on Immobilized Axonin-1 Is Mediated by a Heterophilic Interaction with L1(G4). J Cell Biol. 115:1113-1126.
Kujat, R., Miragall, F., Krause, D., Dermietzel, R. and Wrobel, K. H., 1995. Immunolocalization of the Neural Cell Adhesion Molecule L1 in Non-Proliferating Epithelial Cells of the Male Urogenital Tract. Histochem Cell Biol. 103:311-321.
Kunz, S., Ziegler, U., Kunz, B. and Sonderegger, P., 1996. Intracellular Signaling is Changed After Clustering of the Neural Cell Adhesion Molecules Axonin-1 and NgCAM During Neurite Fasciculation. J Cell Biol. 135:253-267.
L1CAM Mutation Web Page. http://www.rug.nl/umcg/faculteit/disciplinegroepen/medische genetica/hereditarydiseases/l1cam/index. 2009-01-13.
Lagenaur, C. and Lemmon, V., 1987. An L1-Like Molecule, the 8D9 Antigen, Is a Potent Substrate for Neurite Extension. Proc Natl Acad Sci USA. 84:7753-7757.
Liebau, M. C., Gal, A., Superti-Furga, A., Omran, H. and Pohl, M., 2007. L1CAM Mutation in a Boy with Hydrocephalus and Duplex Kidneys. Pediatr Nephrol. 22:1058-1061.
Lindner, J., Rathjen, F. G. and Schachner, M., 1983. L1 Mono- and Polyclonal Antibodies Modify Cell Migration in Early Postnatal Mouse Cerebellum. Nature. 305:427-430.
Lodge, J., Lund, P. and Minchin, S., 2007. Gene Cloning: Principles and Applications. Taylor & Francis Group, New York.
Lüthl, A., Laurent, J. P., Figurov, A., Muller, D. and Schachner, M., 1994. Hippocampal Long-Term Potentiation and Neural Cell Adhesion Molecules L1 and NCAM. Nature. 372:777-779.
Martini, R. and Schachner, M., 1988. Immunoelectron Microscopic Localization of Neural Cell Adhesion Molecules (L1, N-CAM, and Myelin-Associated Glycoprotein) in Regenerating Adult Mouse Sciatic Nerve. J Cell Biol. 106:1735-1746.
Miyahara, R., Tanaka, F., Nakagawa, T., Matsouka, K., Isii, K. and Wada, H., 2001. Expression of Neural Cell Adhesion Molecules (Polysialylated Form of Neural Cell Adhesion Molecule and L1-Cell Adhesion Molecule) on Resected Small Cell Lung Cancer Specimens: in Relation to Proliferation State. J Surg Onocol. 77:49-54.
Montgomery, A. M., Becker, J. C., Siu, C. H., Lemmon, V. P., Cheresh, D. A., Pancook, J. D., Zhao, X. and Reisfeld, R. A., 1996. Human Neural Cell Adhesion Molecule L1 and Rat Homologue NILE are Ligands for Integrin αvβ3. J Cell Biol. 132:475-485.
Moos, M., Tacke, R., Scherer, H., Teplow, D., Früh, K. and Schachner, M., 1988. Neural Adhesion Molecule L1 as a Member of the Immunoglobulin Superfamily with Binding Domains Similar to Fibronectin. Nature. 334:701-703.
Morales, G., Hubert, M., Brümmendorf, T., Treubert, U., Tárnok, A., Schwartz, U. and Rathjen, F. G., 1993. Induction of Axonal Growth by Heterophilic Interactions Between the Cell Surface Recognition Proteins F11 and Nr-CAM/Bravo. Neuron. 11:1113-1122.
New York Times. http://www.nytimes.com/imagepages/2007/08/01/health/adam/8753Corpus callosumofthebrain.html. 2009-01-13.
Nishimura, K., Yoshihara, F., Tojima, T., Ooashi, N., Yoon, W., Mikoshiba, K., Bennett, V.
and Kamiguchi, H., 2003. L1-Dependent Neuritogenesis Involves AnkyrinB That Mediates
L1-CAM Coupling with Retrograde Actin Flow. J Cell Biol. 163:1077-1088.
QIAGEN, 2008a. EZ1 DNA Handbook. http://www1.qiagen.com/HB/BioRobotEZ1Work station_EN_3. 2009-01-13.
QIAGEN, 2008b. QIAxcel DNA Handbook. http://www1.qiagen.com/HB/QIAxcelDNA Kits_EN. 2009-01-13.
Rose, S. P. R., 1995. Cell-Adhesion Molecules, Glucocorticoids and Long-Term-Memory Formation. Trends Neurosci. 18:502-506.
Schuch, U., Lohse, M. J. and Schachner, M., 1989. Neural Cell Adhesion Molecules Influence Second Messenger Systems. Neuron. 3:13-20.
Stallcup, W. B. and Beasley, L., 1985. Involvement of the Nerve Growth Factor-Inducible Large External Glycoprotein (NILE) in Neurite Fasciculation in Primary Cultures of Rat Brain. Proc Natl Acad Sci USA. 82:1276-1280.
Thies, A., Schachner, M., Moll, I., Berger, J., Schulze, H. J., Brunner, G. and Schumacher, U., 2002. Overexpression of the Cell Adhesion Molecule L1 is Associated with Metastasis in Cutaneous Malignant Melanoma. Eur J Cancer. 38:1708-1716.
Thor, G., Probstmeier, R. and Schachner, M., 1987. Characterization of the Cell Adhesion Molecules L1, N-CAM and J1 in the Mouse Intestine. EMBO J. 6:2581-2586.
Williams, E. J., Walsh, F. S. and Doherty, P., 1994. The Production of Arachidonic Acid Can Account for Calcium Channel Activation in the Second Messenger Pathway Underlying Neurite Outgrowth Stimulated by NCAM, N-Cadherin, and L1. J Neurochem. 62:1231-1234. Wong, E. V., Schaefer, W., Landreth, G. and Lemmon, V., 1996a. Casein Kinase II Phosphorylates the Neural Cell Adhesion Molecule L1. J Neurochem. 66:779-786.
Wong, E. V., Schaefer, A. W., Landreth, G. and Lemmon, V., 1996b. Involvement of p90rsk in
Neurite Outgrowth Mediated by the Cell Adhesion Molecule L1. J Biol Chem. 271:18217-18223.
Wood, P. M., Schachner, M. and Bunge, R. P., 1990. Inhibition of Schwann Cell Myelination
