|Kabaeva, Zhyldyz: Genetic analysis in hypertrophic cardiomyopathy: missense mutations in the ventricular myosin regulatory light chain gene |
Aus der Franz-Volhard-Klinik
am Max-Delbrück-Centrum für molekulare Medizin
der Medizinischen Fakultät Charité
der Humboldt-Universität zu Berlin
zur Erlangung des akademischen Grades
Doctor medicinae (Dr. med.)
vorgelegt der Medizinischen Fakultät Charité
der Humboldt-Universität zu Berlin
Dekan / Dean: Prof. Dr. Joachim W. Dudenhausen
Prof. Dr. med. Karl Josef Osterziel
Prof. Dr. med. Andreas Mügge
Prof. Dr. med. Hans-Peter Vosberg
Datum der Promotion / Date of the defence 11.11.2002
Hypertrophic cardiomyopathy (HCM) is a heart disorder characterized by unexplained ventricular myocardial hypertrophy and a high risk of sudden cardiac death. The disease is inherited as an autosomal-dominant trait. Nine disease-causing genes have been described all encoding for sarcomeric proteins. Mutations in the ventricular myosin essential (ELC) and regulatory (RLC) light chain genes are responsible approximately for 1% and 1 - 7% of all HCM cases, respectively. Limited data are available on the disease course and prognosis in HCM caused by mutations in these genes. Therefore, the present study was aimed to analyse the ELC and RLC genes for disease-causing mutations in a group of clinically well-characterized HCM patients. Further purpose was to assess whether the detected mutations are associated with malignant or benign phenotype in the respective families.
Methods: 71 unrelated patients with HCM and 14 family members were evaluated using physical examination, ECG and echocardiography. DNA was extracted from blood lymphocytes. Screening of the 6 exons of the ELC gene and the 7 exons of the RLC gene was done by using PCR and single strand conformation polymorphism analysis (SSCP). Samples with aberrant band patterns were directly sequenced.
Results: Systematic analysis revealed no mutation in the ELC gene but two disease-associated mutations leading to an amino acid exchange in the RLC gene. The first mutation was found in exon 2 of the RLC gene: a G>A nucleotide substitution at position c.64 caused a replacement of glutamic acid by lysine at codon 22. The second mutation was in exon 4 of the RLC gene: a G>A substitution at nucleotide c.173 led to a change of arginine to glutamine at codon 58. Both mutations affected highly conserved amino acids and were located in the amino terminal half of the RLC close to the putative phosphorylation and calcium-binding sites. They also changed overall electrical charge of this protein region. The Glu22Lys mutation was identified in seven individuals of family K and was associated with moderate septal hypertrophy, a late onset of clinical manifestation, benign disease course, and good prognosis. The mutation Arg58Gln showed also moderate septal hypertrophy, but, in contrast, it was associated with an early onset of clinical manifestation and premature sudden cardiac death in family B.
Additionally, a number of sequence differences from reference genomic sequences, one silent mutation, and two single nucleotide polymorphisms (SNPs) were identified while screening the ELC and RLC genes. Detected SNPs did not cause an amino acid exchange and did not affect splicing process proceeding from their localisation.
Conclusions: Two missense mutations were identified in the ventricular myosin regulatory light chain gene and associated with either benign or malignant HCM phenotypes. These findings show that genotyping could give valuable information for risk stratification, genetic counselling, and treatment strategies in hypertrophic cardiomyopathy.
Genetics, Cardiomyopathy, Hypertrophy, Sudden cardiac death
Die Hypertrophe Kardiomyopathie (Hypertrophic Cardiomyopathy, HCM) ist eine Erkrankung des Herzens, die durch eine Hypertrophie des Myokards und einem erhöhten Risiko für den plöztlichen Herztod charakteriziert ist. Die Erkrankung wird autosomal-dominant vererbt. Neun HCM-assozierte Genen wurden bisher beschrieben, die alle für Sarkomer-Proteine kodierend. Mutationen in den Genen für die essentielle (ELC) und regulatorische (RLC) leichte Myosin-Kette sind für ca. 1% bzw. 1-7% aller HCM-Fälle verantwortlich. Bisher gibt es nur wenige Informationen zum Krankheitsverlauf und zur Prognose bei HCM-Formen, die durch Mutationen in diesen Genen verursacht werden. Ziel dieser Studie war daher, das ELC- bzw. RLC-Gen in einem Kollektiv klinisch gut charakterisierter HCM-Patienten hinsichtlich möglicher krankheitsverursachender Mutationen zu analysieren. Darüber hinaus sollte untersucht werden, ob die hier identifizierten Mutationen mit einem malignen bzw. benignen Phänotyp assoziiert sind.
Methoden: 71 unverwandete Patienten mit primärer HCM wurden mittels körperlicher Untersuchung, EKG und Echokardiographie evaluiert. Die aus Blutlymphozyten extrahierte DNA wurde mittels exonspezifischer PCR-Amplifikation und Single-strand-conformation-polymorphism (SSCP) Analyse auf Mutationen in den 6 Exons des ELC- und 7 Exons des RLC-Gens untersucht. Proben mit auffälligen Bandenmustern wurden direkt sequenziert.
Ergebnisse: Die systematische Analyse ergab zwei krankheitsassoziierte Mutationen im RLC-Gen, die zu einem Aminosäurenaustausch führen. Im ELC-Gen wurden keine Mutationen gefunden. Die erste Mutation im RLC-Gen ist ein G zu A-Basenaustausch an Position c.64 im Exon 2, der zu einem Austausch von Glutamat durch Lysin im Codon 22 führt. Die zweite Variante verursacht eine Argininsubstitution durch Glutamin im Codon 58 aufgrund eines Basenpaaraustausches an Position c.173 im Exon 4 (G zu A). Beide Mutationen betreffen hoch-konservierte Aminosäuren in der amino-terminalen Domäne des RLC in der Nähe von möglichen Phosphorylierungs- bzw. Kalcium-Bindungsstellen. Zusätzlich wird die elektrische Ladung dieser Proteinregion durch den Aminosäurenaustausch verändert. Die Glu22Lys-Mutationen konnte in sieben Individuen der Familie K identifiziert werden und ist mit einer geringen septalen Hypertrophie, einer späten klinischen Manifestation sowie einem benignen Verlauf und einer guten Prognose assoziiert. Die Arg58Gln-Mutation ist ebenfalls mit einer moderaten Septumhypertrophie aber mit einem frühen Krankheitsbeginn und einem vorzeitigen Auftreten eines plötzlichen Herztodes in der Familie B assoziiert.
Zusätzlich wurden mehrere Abweichungen von der Referenz-Sequenz, eine stumme Mutation sowie zwei Single Nucleotide Polymorphisms (SNPs) während des Screenings in beiden Genen identifiziert. Die SNPs verursachen keinen Aminosäureaustausch und beeinflussen nicht den Spleißvorgang, soweit dies durch ihre Lokalisation vorhersagbar ist.
Schlussfolgerung: Zwei missense Mutationen konnten in der regulatorischen leichten Myosinkette identifiziert und sowohl mit einem benignen als auch einem malignen HCM-Phänotyp assoziiert werden. Diese Ergebnisse zeigen, dass die Genotypisierung wertvolle Informationen für die Risikostratifizierung, die genetische Beratung sowie für Therapiestrategien in der Hypertrophe Kardiomyopathie liefern kann.
Genetik, Kardiomyopathie, Hypertrophie, plötzlicher Herztod
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Table of Contents
|Front page||Genetic analysis in hypertrophic cardiomyopathy: missense mutations in the ventricular myosin regulatory light chain gene|
|1.1||Molecular genetics and pathogenesis of HCM|
|1.2||The ventricular myosin regulatory and essential light chains|
|1.3||Clinical features and diagnosis of HCM|
|1.4||Genotype-phenotype correlation studies|
|1.5||Aims of the present study|
|2||Materials and methods|
|2.2.2||Preparation of genomic DNA|
|2.2.3||Amplification of coding exons of MYL2 and MYL3|
|2.2.4||Single strand conformation polymorphism analysis|
|2.2.5||Automated DNA sequencing|
|2.2.6||Restriction fragment length polymorphism analysis|
|2.2.7||Agarose gel electrophoresis|
|2.3||Devices and Chemicals|
|3.2||Genetic variants in human MYL2 and MYL3|
|3.2.1||Identification of the Glu22Lys mutation in family K|
|3.2.2||Identification of the Arg58Gln mutation in family B|
|3.2.3||Localization of the mutations in highly conserved RLC regions|
|3.2.4||Clinical features of family K with the Glu22Lys mutation|
|3.2.5||Clinical features of family B with the Arg58Gln mutation|
|3.2.6||The c.420C>T (Phe140Phe) silent mutation in MYL3|
|3.2.7||Single nucleotide polymorphisms in MYL2|
|3.2.8||Genomic sequence differences|
|4.1||Patient cohort and screening approach|
|4.2||The Glu22Lys and Arg58Gln mutations in MYL2|
|4.4||Possible functional implications of the Glu22Lys and Arg58Gln mutations|
|Abbreviations||List of abbreviations|
Table of Tables
|Table 1.1.||Sarcomeric proteins and genes responsible for HCM|
|Table 1.2.||Mutations and prognosis in HCM|
|Table 2.1.||Oligonucleotide primers used to amplify coding exons of MYL2 and MYL3|
|Table 2.2.||Optimised parameters of PCR protocols used to amplify coding exons of MYL2 and MYL3|
|Table 2.3.||Composition of 25 µl PCR mix for one sample|
|Table 2.4.||SSCP conditions used to screen MYL2 and MYL3|
|Table 2.5.||Composition of SSCP gels used to screen MYL2 and MYL3|
|Table 2.6.||Silver staining protocol used to visualize DNA on a SSCP gel|
|Table 2.7.||Components of Dye Primer Chemistry kit|
|Table 2.8.||Agarose gel composition|
|Table 3.1.||Clinical features of HCM patients screened in the present study|
|Table 3.2.||GenBank accession numbers of the reference sequences used in the present study|
|Table 3.3.||Clinical features of genetically affected individuals of families K and B|
|Table 4.1.||The known HCM associated mutations in the RLC and ELC genes|
Table of Figures
|Figure 1.1.||Microscopic structure of heart muscle. A) Myocardium as seen under the light microscope. Myocytes contain a centrally located nucleus and are connected across intercalated disks. B) Myocardial cell reconstituted from electron micrographs. Each myocyte is composed of multiple parallel fibrils. Each fibril is composed of serially connected sarcomeres (N, nucleus). C) Sarcomere from a myofibril, with diagrammatic representation of myofilaments. Thick filaments (1.5 µm long, composed of myosin) from the A band, and thin filaments (1µm long, composed primarily of actin) extend from the Z line through the I band into the A band. The overlapping of thick and thin filaments is seen only in the A band. D) Cross sections of the sarcomere indicate the specific lattice arrangements of the myofilaments. In the center of the sarcomere only the thick, or myosin, filaments arranged in a hexagonal array are seen. In the distal portions of the A band, both thick and thin, or actin, filaments are found, with each thick filament surrounded by six thin filaments. In the I band only thin filaments are present. From ref. 20.|
|Figure 1.2.||Schematic diagram of sarcomere organisation and contraction process. The thin filament is made up of actin, the troponin complex (T,C and I) and -tropomyosin. The thick filament is composed of myosin heavy and light chains. The sarcomere produces muscle contraction by sliding of myofilaments: the myosin heads interact with actin and pull it towards the center of the sarcomere resulting in shortening of the sarcomere. From ref. 2.|
|Figure 1.3.||A) Schematic representation of a myosin molecule constituting the thick filaments of the sarcomere. The myosin molecule is a hexamer consisting of two heavy chains (orange), two essential light chains (blue) and two regulatory light chains (yellow). The myosin heavy chains are dimerized through their coiled-coil tails. Adapted from ref. 22.|
B) Three-dimensional (crystal) structure of a chicken skeletal myosin head. The catalytic and light-chain-binding domains are indicated. The heavy chain is shown in red, green and blue. The essential (yellow) and regulatory (purple) light chains wrap around the heavy chain -helix (blue). Adapted from ref. 10 and 30.
|Figure 2.1.||Schematic representation of the approach for mutation detection undertaken in the present study. DNA, deoxyribose nucleic acid; PCR, polymerase chain reaction; SSCP, single strand conformation polymorphism.|
|Figure 3.1.||Schematic representation of the ventricular myosin regulatory light chain gene (MYL2) and the ventricular myosin essential light chain gene (MYL3). Boxes represent exons, light shaded boxes represent coding DNA of the gene; mutations found in this study are dark shaded; variants with asterisk indicate single nucleotide polymorphisms and the silent mutation; variants without asterisk indicate differences from the reference genomic DNA sequence. Numbering of the genetic variants was performed according to ref. 65. A) Location of two missense mutations, SNPs, and sequence differences in MYL2. B) Location of a silent mutation and sequence differences in MYL3.|
|Figure 3.2.||PCR, SSCP analysis and sequencing of exon 2 of MYL2. A) A 4% agarose gel loaded with 317-bp PCR products of exon 2. M, VIII-DNA ladder; Pr II-3, proband II-3 of family K; 1865 and 1743, other HCM patients; 141 and 142, control individuals; N, negative control. B) Partial SSCP gel. SSCP analysis of proband II-3 of family K (Pr II-3) revealed an aberrant band pattern, which has one additional band (indicated by asterisks) in comparison with patterns shown by neighbouring HCM samples (1438,1434, 1431, and 1427). C) Partial sequence electropherograms of exon 2 of the proband of family K and an individual with the wild type sequence. The proband's electropherogramm showed two typical overlapping peaks at nucleotide c.64: a black peak for guanine on the non-mutated allele and a green peak for adenine on the mutated allele. This G-to-A substitution caused a change of glutamic acid to lysine at codon 22. By contrast, the wild type sequence is homozygous for guanine at position c.64.|
|Figure 3.3.||RFLP analysis of exon 2 of MYL2. A) Schematic representation of restriction sites of Taq I. Two normal S1 and S2 restriction sites produce DNA fragments of 174, 78, and 65 bp. The Glu22Lys mutation removes the S2 restriction site resulting in an abnormal fragment of 252 bp. B) The picture of a 4% agarose gel loaded with restriction fragments of the proband of family K (Pr II-3) and controls (221, 222, 224, 225, and 226). The abnormal digestion fragment of 252 bp due to the Glu22Lys mutation is present in the proband of family K but absent in controls. Lane M contains 125-bp DNA ladder. bp, base pairs; U, undigested PCR product of exon 2.|
|Figure 3.4.||Pedigree of family K and results of RFLP analysis on available family members. Upper panel: Pedigree. Black symbols represent clinically affected patients; white symbols, clinically unaffected individuals; grey symbol, individuals with uncertain phenotype; symbols with plus sign above, genetically affected individuals; symbols with minus sign above, genetically unaffected individuals; and symbols with diagonal slash, deceased individuals. Proband II-3 (patient 1853) is indicated by arrow. Squares, males; circles, females. Lower panel: Identification of the Glu22Lys mutation in family members by RFLP analysis with Taq I. A picture of a 4% agarose gel loaded with restriction digests. The abnormal 252-bp fragment is present in family members II-3, III-2, III-3, II-5, III-5, IV-1, and IV-2 indicating the presence of the Glu22Lys mutation. Lane U contains undigested amplification product of MYL2 exon 2 of 317 base pairs (bp). Lane M contains 125-bp DNA ladder with sizes of bands shown on the right side of the gel picture. Sizes of the digestion products are shown on the left side of the gel picture.|
|Figure 3.5.||SSCP analysis and sequencing of exon 2 of MYL2. A) SSCP analysis. The aberrant band pattern observed in proband II-2 of family B (Pr II-2) was absent in controls (34, 33, 31, 23, and 21) and contains three clear additional bands, which are indicated by asterisks. NBP, normal band pattern; ABP, aberrant band pattern. B) Sequence electropherograms of proband II-2 of family B, the proband's mother, and a control individual. Proband II-2 has two overlapping peaks at position c.173 with a black peak corresponding to guanine on the non-mutated allele and a green peak for adenine on the mutated allele. A G-to-A heterozygous substitution resulted in a replacement of arginine by glutamine at codon 58. By contrast, the Arg58Gln mutation was absent in a control and the proband's mother.|
|Figure 3.6.||Pedigree of family B and results of SSCP analysis. Upper panel: Pedigree. Black symbols represent clinically affected patients; white symbols, clinically unaffected individuals; symbols with plus sign above, genetically affected individuals; symbols with minus sign above, genetically unaffected individuals; symbols with diagonal slash, deceased individuals; squares, males; and circles, females. The proband (patient 1555) is indicated by arrow. SCD, sudden cardiac death. Lower panel: SSCP gel. Proband II-2 showed an aberrant mobility pattern absent in her mother (I-2) and control (C).|
|Figure 3.7.||Amino acid alignment across species and RLC isoforms. The Glu22Lys and Arg58Gln mutations affect the highly conserved amino acids, suggesting the essentiality of these residues for normal protein function. RLCs from the same muscle type show the highest sequence homology. This indicates that functional properties of a protein are determined by its amino acid sequence. Glu and E, glutamic acid; Lys and K, lysine; Arg and R, arginine; Gln and Q, glutamine.|
|Figure 3.8.||Localization of the Glu22Lys and Arg58Gln mutations in the RLC sequence (A) and three-dimensional structure (B). The putative phosphorylation site at serine-15 (A) and calcium-binding site at residues 37-48 (A, B) are highlighted. As shown, the Glu22Lys mutation is located close to the RLC phosphorylation site, moreover it is in the -helix flanking the calcium-binding loop. The Arg58Gln mutation is in the -helix, which flanks the calcium-binding loop from the other side. Adapted from ref. 29.|
|Figure 3.9.||PCR, SSCP analysis and sequencing of exon 4 of MYL3. A) A 4% agarose gel loaded with PCR products of exon 4. The amplified fragments were approximately 400 base pairs (bp) long. Lane M contains VIII-DNA ladder. 1635, a patient carrying the c.420C>T polymorphism; 1781, 1782, and 1783, other HCM patients. B) SSCP analysis of patient 1635 revealed an aberrant band pattern, which has an additional band (indicated by asterisk) in comparison with patterns shown by neighbouring HCM patients 1855, 1853, and 1592. C) Partial sequence of patient 1635 showing the heterozygous c.420C>T silent mutation and of an individual homozygous for the wild type MYL3 allele.|
|Figure 3.10.||Partial sequence electropherograms of intron 4 of MYL2 showing genetic variants at position g.8393. A) DNA sequence of sample 1595 with guanine at nucleotide position g.8393 on both alleles. B) DNA sequence of patient 1819 showing the presence of the homozygous g.8393G>A polymorphism. C) DNA sequence of patient 1811 showing the presence of the heterozygous g.8393G>A polymorphism.|
|Figure 3.11.||Partial sequence electropherograms of intron 5 of MYL2 showing genetic variants at position g.8580. A) DNA sequence of patient 1707 showing the homozygous g.8580C>T polymorphism. B) DNA sequence of wild type sample 1584 with guanine at position g.8580 on both alleles. C) DNA sequence of patient 1594 showing the presence of the g.8580C>T polymorphism on one allele and the g.8580C>A polymorphism on the other allele. D) DNA sequence of patient 1782 showing the presence of the heterozygous g.8580C>T polymorphism.|
|Figure 3.12.||Partial sequence alignment of HCM samples, control and the reference genomic DNA and cDNA sequences. Ref gDNA, the reference genomic DNA sequence; HCM, self-generated DNA sequence of a HCM patient; control, self-generated DNA sequence of a control individual; cDNA, the reference cDNA sequence. A) Partial sequence alignment of intron 1 of MYL2. Three identified differences, g.1277A>G, g.1278G>A and g.1291T>C, are highlighted. B) Partial sequence alignment of exon 4 of MYL2 with the c.240A>T difference highlighted.|
|Figure 3.13.||Partial sequence alignment of HCM samples, control and the reference genomic DNA and cDNA. Ref gDNA, the reference genomic DNA sequence; HCM, self-generated DNA sequence of a HCM patient; control, self-generated DNA sequence of a control individual; cDNA, the reference cDNA sequence; RLC, RLC amino acid sequence translated on the basis of the reference cDNA sequence. A) Sequence alignment of exon 3 and a donor splice site of intron 3 of MYL2. The c.169C>G difference is highlighted. B) Sequence alignment of exon 4 of MYL2. The deletion of adenine and guanine (g.7488_7489delAG) at the acceptor splice site of exon 4 is highlighted. The actual splice site is shown three nucleotides upstream. C) Exon 3 and 4 of MYL2 are aligned together to show that the self-generated sequences are in agreement with the reference cDNA but not with the genomic DNA sequences.|
|Figure 3.14.||RFLP analysis of exon 3 of MYL2, which confirmed the presence of the c.169C>G difference in all of 71 HCM-patients and 100 control individuals. A) Schematic drawing of the restriction sites of Sty I. According to the MYL2 genomic DNA reference sequence, an amplified product of exon 3 is supposed to have only one restriction site (S1), which gives rise to two digests of 35 and 235 base pairs (bp) each. But in the presence of the c.169C>G difference, it has two restriction sites (S1 and S2), which produce three restriction fragments of 35 bp, 92 bp, and 143 bp each. B) A picture of 4% agarose gel loaded with digests. The presence of three restriction fragments of 35, 92, and 143 bp but not two of 35 and 235 confirms the presence of the c.169C>G difference and of an error in the genomic DNA reference sequence. The same band pattern as shown on this gel was observed in all 71 patients and 100 controls. Lane U contains undigested amplification product of exon 3 of 270 base pairs. Lane M contains 125-bp DNA ladder.|
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