Founder mutations in hypertrophic cardiomyopathy patients in the Netherlands.

In this part of a series on cardiogenetic founder mutations in the Netherlands, we review the Dutch founder mutations in hypertrophic cardiomyopathy (HCM) patients. HCM is a common autosomal dominant genetic disease affecting at least one in 500 persons in the general population. Worldwide, most mutations in HCM patients are identified in genes encoding sarcomeric proteins, mainly in the myosin-binding protein C gene (MYBPC3, OMIM #600958) and the beta myosin heavy chain gene (MYH7, OMIM #160760). In the Netherlands, the great majority of mutations occur in the MYBPC3, involving mainly three Dutch founder mutations in the MYBPC3 gene, the c.2373_2374insG, the c.2864_2865delCT and the c.2827C>T mutation. In this review, we describe the genetics of HCM, the genotype-phenotype relation of Dutch founder MYBPC3 gene mutations, the prevalence and the geographic distribution of the Dutch founder mutations, and the consequences for genetic counselling and testing. (Neth Heart J 2010;18:248-54.).

Defective complex I assembly due to C20orf7 mutations as a new cause of Leigh syndrome.

BACKGROUND: Leigh syndrome is an early onset, progressive, neurodegenerative disorder with developmental and motor skills regression. Characteristic magnetic resonance imaging abnormalities consist of focal bilateral lesions in the basal ganglia and/or the brainstem. The main cause is a deficiency in oxidative phosphorylation due to mutations in an mtDNA or nuclear oxidative phosphorylation gene. METHODS AND RESULTS: A consanguineous Moroccan family with Leigh syndrome comprise 11 children, three of which are affected. Marker analysis revealed a homozygous region of 11.5 Mb on chromosome 20, containing 111 genes. Eight possible mitochondrial candidate genes were sequenced. Patients were homozygous for an unclassified variant (p.P193L) in the cardiolipin synthase gene (CRLS1). As this variant was present in 20% of a Moroccan control population and enzyme activity was only reduced to 50%, this could not explain the rare clinical phenotype in our family. Patients were also homozygous for an amino acid substitution (p.L159F) in C20orf7, a new complex I assembly factor. Parents were heterozygous and unaffected sibs heterozygous or homozygous wild type. The mutation affects the predicted S-adenosylmethionine (SAM) dependent methyltransferase domain of C20orf7, possibly involved in methylation of NDUFB3 during the assembly process. Blue native gel electrophoresis showed an altered complex I assembly with only 30-40% of mature complex I present in patients and 70-90% in carriers. CONCLUSIONS: A new cause of Leigh syndrome can be a defect in early complex I assembly due to C20orf7 mutations.

A single hERG mutation underlying a spectrum of acquired and congenital long QT syndrome phenotypes.

The long QT syndrome (LQTS) is a multi-factorial disorder that predisposes to life-threatening arrhythmias. Both hereditary and acquired subforms have been identified. Here, we present clinical and biophysical evidence that the hERG mutation c.1039 C>T (p.Pro347Ser or P347S) is responsible for both the acquired and the congenital phenotype. In one case the genotype remained silent for years until the administration of several QT-prolonging drugs resulted into a full-blown phenotype, that was reversible upon cessation of these compounds. On the other hand the mutation was responsible for a symptomatic congenital LQTS in a Dutch family, displaying a substantial heterogeneity of the clinical symptoms. Biophysical characterization of the p.Pro347Ser potassium channels using whole-cell patch clamp experiments revealed a novel pathogenic mechanism of reciprocal changes in the inactivation kinetics combined with a dominant-negative reduction of the functional expression in the heterozygous situation, yielding a modest genetic predisposition for LQTS. Our data show that in the context of the multi-factorial aetiology underlying LQTS a modest reduction of the repolarizing power can give rise to a spectrum of phenotypes originating from one mutation. This observation increases the complexity of genotype-phenotype correlations in more lenient manifestations of the disease and underscores the difficulty of predicting the expressivity of the LQTS especially for mutations with a more subtle impact such as p.Pro347Ser.

Methods in molecular cardiology: DHPLC mutation detection analysis.

An increasing number of mutations have been identified in genes involved in cardiac disorders which has led to novel insights in the pathophysiology of inherited cardiac diseases. As a result of these findings, techniques specialised in automated high-throughput analysis are implemented to handle the increasing number of diagnostic genetic requests. Denaturing high-performance liquid chromatography (DHPLC) is one such novel technique that fulfils the criteria of speed, sensitivity and accuracy. This issue focuses on the basic principle of the technique and illustrates how genetic alterations can be identified.

Pearson syndrome and the role of deletion dimers and duplications in the mtDNA.

Pearson syndrome is an often fatal multisystem disease associated with mitochondrial DNA rearrangements. Here we report a patient with a novel mtDNA deletion of 3.4 kb ranging from nucleotides 6097 to 9541 in combination with deletion dimers. The mutation percentage in different tissues (blood, muscle and liver) varied between 64% and 95%. After a remission period of about a year, the patient suddenly died at the age of 3 years owing to a severe lactic acidosis. A second patient with a previously reported deletion of 8 kb and a milder phenotype was found to have mitochondrial duplications and died at the age of 10 years. From these data and data from previous reports, we hypothesize that duplications might be beneficial in the clinical course of the disease and in life expectancy.

Novel KCNQ1 and HERG missense mutations in Dutch long-QT families.

Congenital long QT syndrome (cLQTS) is electrocardiographically characterized by a prolonged QT interval and polymorphic ventricular arrhythmias (torsade de pointes). These cardiac arrhythmias may result in recurrent syncopes, seizure, or sudden death. LQTS can occur either as an autosomal dominant (Romano Ward) or as an autosomal recessive disorder (Jervell and Lange-Nielsen syndrome). Mutations in at least five genes have been associated with the LQTS. Four genes, encoding cardiac ion channels, have been identified. The most common forms of LQTS are due to mutations in the potassium-channel genes KCNQ1 and HERG. We have screened 24 Dutch LQTS families for mutations in KCNQ1 and HERG. Fourteen missense mutations were identified. Eight of these missense mutations were novel: three in KCNQ1 and five in HERG. Novel missense mutations in KCNQ1 were Y184S, S373P, and W392R and novel missense mutations in HERG were A558P, R582C, G604S, T613M, and F640L. The KCNQ1 mutation G189R and the HERG mutation R582C were detected in two families. The pathogenicity of the mutations was based on segregation in families, absence in control individuals, the nature of the amino acid substitution, and localization in the protein. Genotype-phenotype studies indicated that auditory stimuli as trigger of cardiac events differentiate LQTS2 and LQTS1. In LQTS1, exercise was the predominant trigger. In addition, a number of asymptomatic gene defect carriers were identified. Asymptomatic carriers are still at risk of the development of life-threatening arrhythmias, underlining the importance of DNA analyses for unequivocal diagnosis of patients with LQTS.

Auditory stimuli as a trigger for arrhythmic events differentiate HERG-related (LQTS2) patients from KVLQT1-related patients (LQTS1).

OBJECTIVE: This study was performed to identify a possible relationship between genotype and phenotype in the congenital familial long QT syndrome (cLQTS). BACKGROUND: The cLQTS, which occurs as an autosomal dominant or recessive trait, is characterized by QT-interval prolongation on the electrocardiogram and torsade de pointes arrhythmias, which may give rise to recurrent syncope or sudden cardiac death. Precipitators for cardiac events are exercise or emotion and occasionally acoustic stimuli. METHODS: The trigger for cardiac events (syncope, documented cardiac arrhythmias, sudden cardiac death) was analyzed in 11 families with a familial LQTS and a determined genotype. RESULTS: The families were subdivided in KVLQT1-related families (LQTS1, n = 5) and HERG (human ether-a-gogo-related gene)-related families (LQTS2, n = 6) based on single-strand conformation polymorphism analysis and sequencing. Whereas exercise-related cardiac events dominate the clinical picture of LQTS1 patients, auditory stimuli as a trigger for arrhythmic events were only seen in LQTS2 patients. CONCLUSIONS: Arrhythmic events triggered by auditory stimuli may differentiate LQTS2 from LQTS1 patients.

The long QT syndrome: a novel missense mutation in the S6 region of the KVLQT1 gene.

The Romano Ward long QT syndrome (LQTS) has an autosomal dominant mode of inheritance. Patients suffer from syncopal attacks often resulting in sudden cardiac death. The main diagnostic parameter is a prolonged QT(c) interval as judged by electro-cardiographic investigation. LQTS is a genetically heterogeneous disease with four loci having been identified to date: chromosome 11p15.5 (LQT1), 7q35-36 (LQT2), 3p21-24 (LQT3) and 4q25-26 (LQT4). The corresponding genes code for potassium channels KVLQT1 (LQT1) and HERG (LQT2) and the sodium channel SCN5A (LQT3). The KVLQT1 gene is characterized by six transmembrane domains (S1-S6), a pore region situated between the S5 and S6 domains and a C-terminal domain accounting for approximately 60% of the channel. This domain is thought to be co-associated with another protein, viz. minK (minimal potassium channel). We have studied a Romano Ward family with several affected individuals showing a severe LQTS phenotype (syncopes and occurrence of sudden death). Most affected individuals had considerable prolongations of QT(c). By using haplotyping with a set of markers covering the four LQT loci, strong linkage was established to the LQT1 locus, whereas the other loci (LQT2, LQT3 and LQT4) could be excluded. Single-strand conformation polymorphism analysis and direct sequencing were used to screen the KVLQT1 gene for mutations in the S1-S6 region, including the pore domain. We identified a Gly-216-Arg substitution in the S6 transmembrane domain of KVLQT1. The mutation was present in all affected family members but absent in normal control individuals, providing evidence that the mutated KVLQT1-gene product indeed caused LQTS in this family. The mutated KVLQT1-gene product thus probably results in a dominant negative suppression of channel activity.

A deletion of 1.6 kb proximal to the CGG repeat of the FMR1 gene causes the clinical phenotype of the fragile X syndrome.

The vast majority of individuals with the fragile X syndrome show expanded stretches of CGG repeats in the 5' non-coding region of FMR1. This expansion coincides with abnormal methylation patterns in that area resulting in the silencing of the FMR1 gene. Evidence is accumulating that this directly causes the fragile X phenotype. Very few other mutations in FMR1, causing the fragile X phenotype have been reported thus far and all concerned isolated cases. We, however, report a family, in which 11 individuals have a deletion of 1.6 kb proximal to the CGG repeat of the FMR1 gene. Although fragile X chromosomes were not detected, all 4 affected males and 2 of the carrier females show characteristics of the fragile X phenotype. Using RT-PCR we could demonstrate that FMR1 is not expressed in the affected males, strongly suggesting that the FMR1 promoter sequences 5' to the CGG repeat are missing. The deletion patients have approximately 45 CGG repeats in their FMR1 gene, though not interspersed by AGG triplets that are usually present in both normal and expanded repeats. It is hypothesized that prior to the occurrence of the deletion, an expansion of the repeat occurred, and that the deletion removed the 5' part of the CGG repeat containing the AGG triplets. Transmission of the deletion through the family could be traced back to the deceased grandfather of the affected males, which supports the hypothesis that the FMR1 gene product is not required for spermatogenesis. Finally, the data provide additional evidence that the fragile X syndrome is a single gene disorder.

RFLP haplotyping and mutation analysis of the phenylalanine hydroxylase gene in Dutch phenylketonuria families.

Restriction fragment length polymorphism haplotyping of mutated and normal phenylalanine hydroxylase (PAH) alleles in 49 Dutch phenylketonuria (PKU) families was performed. All mutant PAH chromosomes identified by haplotyping (n = 98) were screened for eight of the most predominant mutations. Compound heterozygosity was proven in 40 kindreds. Homozygosity was found for the IVS/2nt1 mutation in 5 families, and for the R158Q and IVS10nt546 mutations in one family each. All patients from these families suffer from severe PKU, providing additional proof that these mutations are deleterious for the PAH gene. Genotypical heterogeneity was evident for mutant haplotype 1 (n = 27) carrying the mutations R261Q (n = 12), E280K (n = 4, P281L (n = 1) and unknown (n = 10), and likewise for mutant haplotype 4 (n = 30) carrying the mutations R158Q (n = 13), Y414C (n = 1) and unknown (n = 16). Mutant haplotype 3 (n = 20), in tight association with mutation IVS12nt1, appeared to be in strong linkage disequilibrium (LDE) with its normal counterpart allele (n = 4). Mutant haplotype 6 (n = 4), in tight association with the IVS10nt546 mutation, showed moderate LDE with its counterpart allele (n = 1). The distribution of the mutant PAH haplotypes 1, 3 and 4 among the Dutch PKU population resembles that in other Northern and Western European countries, but it is striking that mutant haplotype 2 and its associated mutation R408W is nearly absent in The Netherlands, in strong contrast to its neighbouring countries.

[Phenylketonuria as a model system for DNA diagnosis of hereditary disorders]. / Fenylketonurie als modelsysteem voor DNA-diagnostiek van erfelijke aandoeningen.

Phenylketonuria (PKU), due to a defect in phenylalanine hydroxylase (PAH), is presented as a model system for computer-aided DNA diagnosis of genetic diseases. Eight different restriction fragment length polymorphism (RFLP) markers have been localized within the introns of the 90 kb PAH gene (located on chromosome 12). These RFLPs can be combined in 384 different ways and each combination has been defined as a particular haplotype. A special computer program has been developed to calculate the possible haplotype combinations in a PKU core family (index patient and parents), with the goal to derive unambiguously both the PAH and PKU alleles. Taking into account that participation of other members of the family (grandparents or brothers/sisters) is sometimes necessary, haplotyping by itself is sufficient to establish (or exclude) the PKU status of an individual in approximately eight out of ten PKU families.

Rate of recombination of chromosomes 21 in parents of children with Down syndrome.

To test the hypothesis of reduced chiasma frequency causing nondisjunction during meiosis, we examined 34 Down syndrome patients and their parents. Chromosomal polymorphisms and RFLP markers were used to trace the parental origin as well as the frequency of recombination of chromosomes 21. In all but one case, the parental origin and the meiotic stage of nondisjunction could be established by either technique. In 11 cases recombination could be deduced to have taken place during meiosis in the parent who contributed the extra chromosome 21. Because of the underestimation which is inherent in the methods used, these results do not seem to support the chiasma theory.

Human gametes and zygotes studied by nonradioactive in situ hybridization.

A nonradioactive in situ hybridization technique was applied to human gametes and abnormally fertilized or developed zygotes. Using haptenized chromosome-specific probes, visualization was obtained using immunocytochemistry to achieve a fluorescent stain on specific hybrids. Using a chromosome 1-specific DNA probe, almost all spermatozoa gave a positive result, i.e., one hybridization signal per cell could be observed. Furthermore, it was possible to identify sperm cells with two spots, suggesting nondisjunction. Two cleavage arrested embryos from different patients showed both: two brightly fluorescent spots and two weaker spots with the same DNA probe. Using a Y-specific DNA probe the percentages of positive spermatozoa from the normal males ranged between 48.1% and 49.1%. In an embryo with four grossly haploid chromosome sets, three fluorescent spots were obtained with the Y-specific DNA probe, indicating the penetration of three spermatozoa.

Distribution of meiotic recombination along nondisjunction chromosomes 21 in Down syndrome determined using cytogenetics and RFLP haplotyping.

Ten families (Down syndrome children and their parents) showing evidence of meiotic recombination between intraparental chromosomes transmitted after nondisjunction were studied. Cytogenetic polymorphisms and a cassette of RFLP markers distributed along chromosome 21 were used to analyze these families to localize the regions of meiotic recombination. Results indicated that only one crossover occurred per meiotic division and that nine of ten nondisjunctions appeared to be of maternal origin. In one family the crossover had taken place in the pericentromeric region, proximal to marker D21S13, which is quite exceptional. A chance of meiotic recombination within region 21q21, flanked by marker D21S72 and the amyloid gene, could be demonstrated in seven of the ten families. Most strikingly, this chance significantly decreased distal to q21, with frequencies of 0.3 and 0.1 in regions q22.2 and q22.3-qter, respectively. It is hypothesized that decreased chiasmata formation in the most distal part of chromosome 21q might promote nondisjunction. Furthermore, data from the ten crossovers made it possible to map provisionally two previously undefined markers, D21S24 and D21S82, to regions q21-qter and q22.1-qter, respectively.

On the origin of recurrent trisomy 21: determination using chromosomal and DNA polymorphisms.

A family is described in which two cases of trisomy 21 occurred in, respectively, a newborn infant and a prenatally diagnosed fetus. Using fluorescent chromosomal polymorphisms, it was established that in both cases the extra chromosome resulted from a first meiotic division error in the mother and that the father contributed the same centromeric region to both children. RFLP-associated probes were used to examine the genetic content of the chromosomes. It was noted that the polymorphism patterns of the chromosomes 21 which both children inherited from their parents were identical for three, but not identical for one of the probes studied. This difference must be the result of recombination. This result is discussed in relation to the suggestion that the increased recurrence rate in mothers with a trisomic child could be due to a reduced recombination rate.

Characterization of the rat gamma-crystallin gene family and its expression in the eye lens.

Rat genomic clones, which together contain all of the rat genomic gamma-crystallin sequences, have been characterized. Five gamma-crystallin genes are located on a contiguous DNA region, 63 X 10(3) base-pairs long. These genes, named (5') gamma 1-1, gamma 1-2, gamma 2-2 and gamma 3-1 (3'), are all oriented head to tail. A sixth gamma-crystallin gene, named the gamma 4-1 gene, could not be linked to the gamma-crystallin gene cluster with our present set of genomic clones. Mapping experiments using single copy sequences which form the extreme 5' or 3' region of the gene cluster showed that, if the gamma 4-1 gene is located on the same chromosome, then it must be separated from the gene cluster by at least 25 X 10(3) base-pairs of DNA. All gamma-crystallin genes have a similar mosaic structure. They contain a large (0.9 X 10(3) to 1.88 X 10(3) base-pairs) intron in the middle of the gene and are further interrupted close to the 5' end of the gene. The length of the first exon varies from about 40 to about 50 base-pairs. The complementary DNA clone pRL-gamma-3 used in this study is a copy of the transcript of the gamma 3-1 gene, while the second complementary DNA clone, pRL-gamma-2, is most likely a copy of the transcript of the gamma 2-1 gene. It is further shown that rat lens messenger RNA protects fragments from the 3' ends of the four other gamma-crystallin genes against degradation by S1 nuclease, hence all six gamma-crystallin genes present in the rat genome must be transcribed in the lens. Repetitive sequences were found to be present between and around the gamma-crystallin genes. Mapping with cloned repetitive sequences showed that three different repeats, designated A, B and C, occur more than once in the gamma-crystallin gene cluster. Repeat C is also found in the gamma 4-1 region. A repetitive region 3' to the gamma 3-1 gene contains members of all three repeat families.

Human lens gamma-crystallin sequences are located in the p12-qter region of chromosome 2.

The human gamma-crystallin genes constitute a multigene family whose members are only expressed in the eye lens. The chromosomal location of these sequences has been determined by screening a panel of human/rodent hybrid cell lines containing overlapping subsets of human chromosomes for the presence of human gamma-crystallin sequences. By correlating these genomic hybridization data with the chromosomal constitution of the somatic cell hybrids, all human gamma-crystallin sequences could be assigned to chromosome 2. The use of human/hamster cell hybrids derived from human Burkitt lymphoma cells carrying a reciprocal translocation between human chromosomes 2 and 8, allowed a further localization of the sequences to the region 2p12-qter.