Genotype-phenotype Correlation Studies Research Articles

Genotyping and haplotyping of CYP2C19 functional alleles on thin-film biosensor chips

Numerous functional polymorphisms in the CYP2C19 gene have been identified; some alleles (e.g. CYP2C19*2 and CYP2C19*3) are associated with poor metabolism of CYP2C19 substrate drugs. Studies have found that the proportion of poor metabolizers, explained by CYP2C19*2 and CYP2C19*3, varies from less than 50% to more than 90% of poor metabolizers. Therefore, phenotype-genotype correlation studies should cover more than CYP2C19*2 and CYP2C19*3. A broader coverage, however, requires an easy-to-use and high-throughput genotyping platform. This broader coverage should also include the recently identified functional allele, CYP2C19*10, which involves a nucleotide change adjacent to the altered nucleotide change in CYP2C19*2. The currently used restriction fragment length polymorphism-based method for genotyping CYP2C19*2 cannot distinguish between CYP2C19*2 and CYP2C19*10. We aim to develop a simple platform that can genotype all CYP2C19 functional alleles. We have developed a thin-film biosensor chip platform to genotype 16 exonic CYP2C19 variants, including two sets of two adjacent single nucleotide polymorphisms and 12 single single nucleotide polymorphisms, using a ligation strategy. We demonstrate that this is a rapid, accurate, and inexpensive method for genotyping CYP2C19 variants using individual's genomic DNA samples. We further demonstrate that this genotyping platform can be used to construct a haplotype structure of the CYP2C19 variants in a population, and to assign a haplotype combination to each individual on the basis of his/her genotype results. This assay can be applied in pharmacogenomic studies in both basic research and clinical laboratories. It is also an ideal technology for pharmacogenomic tests in both developed and developing countries.

Chromosome 11 segmental paternal isodisomy in amniocytes from two fetuses with omphalocoele: new highlights on phenotype–genotype correlations in Beckwith–Wiedemann syndrome

Background: The phenotypic variability in Beckwith–Wiedemann syndrome (BWS) reflects the genetic heterogeneity of the mechanism which by default leads to the deregulation of genes located at 11p15.5. Genotype–phenotype correlation studies...

The Long QT Syndrome

The long QT syndrome (LQTS) is a genetically transmitted cardiac arrhythmia due to ion channel protein abnormalities, which affects the transport of potassium and sodium ions across the cell membrane. Patients with LQTS may present with syncope, seizures or aborted cardiac arrest. LQTS is also an important cause of unexplained sudden cardiac death in the young. The diagnosis of LQTS is generally made on an ECG showing the prolonged QT interval. The establishment of LQTS registry and the discovery of genetic mutations causing LQTS have contributed greatly to the understanding of this condition and have also provided an impetus in understanding of other inherited cardiac arrhythmias. Genotype-phenotype correlation studies have allowed risk stratification of LQTS patients. Life style modification to avoid triggers for malignant cardiac arrhythmias, and the use of beta-blockers, pacemakers and implantable defibrillators, help to treat symptoms and reduce mortality in these patients.

Genetics of congenital heart diseases in syndromic and non-syndromic patients: new advances and clinical implications

Congenital heart defects (CHDs) are the most common birth defects in humans and over the last 20 years significant progress has been made in the understanding of the molecular and genetic determinants of an increasing number of CHDs. Fundamental to this progress has been the contribution of five fields of research: the epidemiological results of the Baltimore-Washington Infant Study (BWIS); the pathogenetic classification introduced by Clark; the Human Genome Project; genotype-phenotype correlation and familial recurrence studies; and transgenic animals. The recently advanced cytogenetic techniques can now detect subtle rearrangements in chromosomes, which may be overlooked by standard methods and, more recently, molecular instruments such as linkage analysis and positional cloning are being used to identify genes causing Mendelian monogenic syndromes with CHDs, such as Holt-Oram, Ellis-van Creveld and Noonan/LEOPARD syndromes. Finally, useful information is yet available with regard to genes causing isolated CHDs in individuals who do not have a genetic syndrome (an example is the mutation of NKX2.5 and GATA4 genes causing atrial septal defect). The future perspectives for the genetics of CHDs will involve three fields of interest: diagnosis; therapy; and prognosis.

Clinical‐etiologic correlation in children with Prader‐Willi syndrome (PWS): An interdisciplinary study

Prader-Willi syndrome (PWS) is a multisystemic disorder caused by the loss of expression of paternally transcribed genes within chromosome 15q11-q13. Most cases are due to paternal deletion of this region; the remaining cases result from maternal uniparental disomy (UPD) and imprinting defects. To better understand the phenotypic variability of PWS, a genotype-phenotype correlation study was performed in 91 children with PWS. Patients were diagnosed by Southern Blot Methylation assay and genetic subtypes were established using FISH and microsatellite analyses. Fifty-nine subjects with deletion (31/28 males/females; mean age 3.86 years), 30 with UPD (14/16 males/females; mean age 3.89 years) and 2 girls with a presumed imprinting defect (mean age 0.43 yrs) were identified. For correlation purposes patients were grouped as "deleted" and "non-deleted." An increased maternal age was found in the UPD group. Four of Holm's criteria were more frequently present in the deleted group: need for special feeding techniques, sleep disturbance, hypopigmentation, and speech articulation defects. Concerning cognitive assessments, only 9.52% of subjects with deletion had Full-Scale IQ (FSIQ) > or =70, while 61.53% of subjects without deletion had FSIQ > or =70. Similar results were found in behavioral measures. Sleep disorders and carbohydrate metabolism were systematically assessed. Polysomnoghaphic studies revealed a higher frequency of central events with desaturations > or =10% in the deleted group (P = 0.020). In summary, the phenotype was significantly different between both groups in certain parameters related to the CNS. These results might be related to the differences in brain gene expression of the genetic subtypes.

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Cri du Chat syndrome

The Cri du Chat syndrome (CdCS) is a genetic disease resulting from a deletion of variable size occurring on the short arm of chromosome 5 (5p-). The incidence ranges from 1:15,000 to 1:50,000 live-born infants. The main clinical features are a high-pitched monochromatic cry, microcephaly, broad nasal bridge, epicanthal folds, micrognathia, abnormal dermatoglyphics, and severe psychomotor and mental retardation. Malformations, although not very frequent, may be present: cardiac, neurological and renal abnormalities, preauricular tags, syndactyly, hypospadias, and cryptorchidism. Molecular cytogenetic analysis has allowed a cytogenetic and phenotypic map of 5p to be defined, even if results from the studies reported up to now are not completely in agreement. Genotype-phenotype correlation studies showed a clinical and cytogenetic variability. The identification of phenotypic subsets associated with a specific size and type of deletion is of diagnostic and prognostic relevance. Specific growth and psychomotor development charts have been established. Two genes, Semaphorin F (SEMAF) and δ-catenin (CTNND2), which have been mapped to the "critical regions", are potentially involved in cerebral development and their deletion may be associated with mental retardation in CdCS patients. Deletion of the telomerase reverse transcriptase (hTERT) gene, localised to 5p15.33, could contribute to the phenotypic changes in CdCS. The critical regions were recently refined by using array comparative genomic hybridisation. The cat-like cry critical region was further narrowed using quantitative polymerase chain reaction (PCR) and three candidate genes were characterised in this region. The diagnosis is based on typical clinical manifestations. Karyotype analysis and, in doubtful cases, FISH analysis will confirm the diagnosis. There is no specific therapy for CdCS but early rehabilitative and educational interventions improve the prognosis and considerable progress has been made in the social adjustment of CdCS patients.

Mutations inRYR1in malignant hyperthermia and central core disease

The RYR1 gene encodes the skeletal muscle isoform ryanodine receptor and is fundamental to the process of excitation-contraction coupling and skeletal muscle calcium homeostasis. Mapping to chromosome 19q13.2, the gene comprises 106 exons and encodes a protein of 5,038 amino acids. Mutations in the gene have been found in association with several diseases: the pharmacogenetic disorder, malignant hyperthermia (MH); and three congenital myopathies, including central core disease (CCD), multiminicore disease (MmD), and in an isolated case of a congenital myopathy characterized on histology by cores and rods. The majority of gene mutations reported are missense changes identified in cases of MH and CCD. In vitro analysis has confirmed that alteration of normal calcium homeostasis is a functional consequence of some of these changes. Genotype-phenotype correlation studies performed using data from MH and CCD patients have also suggested that mutations may be associated with a range of disease severity phenotypes. This review aims to summarize the current understanding of RYR1 mutations reported in association with MH and CCD and the present viewpoint on the use of mutation data to aid clinical diagnosis of these conditions.

Novel Locus for an Inherited Cardiomyopathy Maps to Chromosome 7

Genetic mutations are the most common cause of hypertrophic cardiomyopathy (HCM) and an increasingly recognized cause of dilated cardiomyopathy. Autosomal dominant HCM is caused by mutations in sarcomere proteins; such mutations are not universally present, however, and fail to account for &40% of cases of phenotypic HCM. To add further complexity, other genetic origins can mimic the gross clinical phenotype of HCM, and mutations in sarcomere genes have been demonstrated to cause dilated cardiomyopathy. To explore novel genetic causes of inherited cardiomyopathies, genome-wide linkage analysis was used to study one kindred (4 generations, 32 individuals) with predominant clinical features of left ventricular hypertrophy in addition to cardiac dilation, end-stage heart failure, and sudden death. Of note, histopathology from 2 family members did not demonstrate myocyte disarray and fibrosis, indicating that this phenotype is not typical sarcomere mutation HCM. Direct DNA sequencing was performed on sarcomere genes known to cause HCM and dilated cardiomyopathy, and no mutations were identified. Linkage was then established to a novel locus on chromosome 7 (7p12.1-7q21). A maximum 2-point logarithm of odds score of 4.11 was obtained. Recombination events refine the disease interval between D7S506 and D7S3314, corresponding to a distance of 27.2 megabases. The discovery of a novel genetic locus in this family provides more evidence that molecular pathways leading to inherited cardiac hypertrophy extend beyond the sarcomere. Identification of the causal gene mutation and additional genotype-phenotype correlation studies will provide fundamental insight into mechanisms of cardiac remodeling.

Clinical characteristics of myotonic dystrophy type 1 patients with small CTG expansions

The authors report a genotype-phenotype correlation study in 102 patients with myotonic dystrophy type 1 carrying small CTG repeat expansions. Most patients carrying 50 to 99 CTG repeats were asymptomatic, except for cataracts. Myotonia, weakness, excessive daytime sleepiness, and myotonic discharges at EMG were significantly more present in the patients with 100 to 200 CTG repeats. These findings highlight different outcomes related to the expansion size, even among small CTG expansions.

Does Genotype Predict Phenotype in Rett Syndrome?

Mutations in the X-linked gene encoding the methyl-CpG binding protein MeCP2 are the primary cause of classic and atypical Rett syndrome and have recently been shown to contribute to other neurodevelopmental disorders of varying severity. To determine whether there are molecular correlates to the phenotypic heterogeneity, numerous groups have performed genotype-phenotype correlation studies. These studies have yielded conflicting results, in part because they used different criteria for determining severity and classifying mutations. Evolution of the phenotype with age and variable expressivity arising from individual variability in X-chromosome inactivation patterns are among other reasons the findings varied. Nonetheless, evidence of differences in the phenotypic consequences of specific types of mutations is emerging. This review analyzes the available literature and makes recommendations for future studies.

Should chromosome breakage studies be performed in patients with VACTERL association?

The VACTERL association is characterized as a non-random pattern of defects including at least three of the following cardinal features: vertebral anomalies, anal atresia, cardiovascular malformations, tracheoesophageal fistula, renal and limb anomalies, and is postulated to be a very heterogeneous disorder. These defects can also be seen as part of the Fanconi anemia (FA) spectrum. Although VACTERL with hydrocephaly has clearly been associated with FA, the indication for chromosome breakage studies is not clear in VACTERL without hydrocephaly. We report on three unrelated patients with the VACTERL phenotype and the confirmed diagnosis of FA. Together with the data of 13 similar cases extracted from a European genotype-phenotype correlation study for FA and those from the four reported cases of the literature, we show that (i) in a series of individuals proven to have FA, 5% (13/245) also have the VACTERL phenotype, (ii) all have radial ray anomalies and 12 of these 13 subjects show at least 1 other feature of FA (café au lait spots, growth retardation, microcephaly, dysmorphism), and (iii) the VACTERL phenotype appears to be over represented in the FA complementation groups D1, E, and F. Since the diagnosis of FA is important for genetic counseling and early therapeutic intervention in patients, we conclude that chromosomal breakage studies should be performed, not only in cases of VACTERL with hydrocephaly, but also in cases VACTERL with radial-ray anomalies and especially if the individual has additional FA associated manifestations such as skin pigmentation abnormalities, growth retardation, microcephaly, or microphthalmia.

A genotype-phenotype correlation study in a group of 54 patients with X-linked agammaglobulinemia

X-linked (Bruton's) agammaglobulinemia (XLA) is a rare immunodeficiency caused by a block in B-cell development caused by mutations in the Bruton's tyrosine kinase (BTK) gene. Many aspects of XLA and BTK function remain unresolved; atypical presentations have been reported, and no clear genotype-phenotype correlation has been established. We sought to contribute to the understanding of XLA through the phenotypic and biochemical characterization of a large group of Spanish patients with agammaglobulinemia. We also sought to classify the mutations according to their severity to analyze a genotype-phenotype correlation. Clinical and analytic data were collected from the clinical records. We studied the BTK gene, protein expression, and function, and the findings were correlated with the phenotypic information. Fifty-four patients were given diagnoses of XLA. We identified 38 different mutations in BTK, 26 not described in other patients, and several uncommon clinical phenotypes or analytic characteristics were found. The statistical analysis shows that less severe mutations or minimal detection of protein by means of flow cytometry are associated with decreased severity in clinical and analytic data, demonstrating a clear relation between the type of mutation and the disease expressivity. However, some exemptions to this rule were noted. XLA is a variable disease. Globally, a genotype-phenotype correlation is observed, but individual discrepancies between the severity of the mutation and the clinical and analytic phenotype suggest that other loci or ambient factors significantly influence the disease presentation and evolution.

Genetic testing for risk stratification in hypertrophic cardiomyopathy and long QT syndrome: fact or fiction?

Hypertrophic cardiomyopathy, affecting 1 in 500 persons, is the most common identifiable cause of sudden cardiac death in the young, whereas congenital long QT syndrome, affecting 1 in 5000 persons, is perhaps one of the most common causes of autopsy negative sudden unexplained death. Since May 2004, genetic testing has been available as a clinical diagnostic test for both hypertrophic cardiomyopathy and long QT syndrome. It is now critical to carefully scrutinize the relationships between genotype and phenotype as they pertain to clinical practice. In 1990, the molecular underpinnings of hypertrophic cardiomyopathy were exposed with the identification of a mutation in the MYH7-encoded beta myosin heavy chain. Since then, hundreds of mutations scattered among at least 14 genes confer the pathogenetic substrate for this 'disease of the sarcomere'. In 1995, the discipline of cardiac channelopathies was born with the revelation that mutations in critical cardiac channel genes cause long QT syndrome. Today, hundreds of mutations involving several cardiac channel genes account for approximately 75% of long QT syndrome. Over the past decade, scores of genotype-phenotype correlation studies in both hypertrophic cardiomyopathy and long QT syndrome have been conducted. Genomic medicine has now entered the clinical practice as it pertains to the evaluation and management of both hypertrophic cardiomyopathy and long QT syndrome. The diagnostic utility of genetic testing for both diseases is clearly evident, as well as current limitations. While treatment decisions are certainly influenced by knowing the underlying genotype in long QT syndrome, there seems to be negligible prognostic value associated with particular hypertrophic cardiomyopathy-causing mutations at this time.

Rett syndrome

Nearly 70 reports on Rett syndrome were published in 2004. We have selected 51 articles, including clinical reports, on pathophysiology, genotype-phenotype correlation, and clinical and basic molecular biology studies. These articles explain how mutation of the gene (MECP2) for methyl-CpG-binding protein 2 causes the particular disorders of Rett syndrome, and also induces other neurodevelopmental disorders, clarifying the situation for future studies. The role of X-chromosome inactivation has been clarified in animal experiments. New isoforms of MeCP2 have been discovered and its functional characteristics are under research. Understanding of the influence of the MECP2 mutation on other neurodevelopmental disorders has increased. However, there is no apparent progress in neurophysiological studies. Clinical studies included the pathophysiology of stereotyped movement, and cardiac and respiratory disturbances, and there were four therapeutic trials including one for epilepsy. For genotype-phenotype correlation the role of X-chromosome inactivation was looked at and its basic mechanisms were studied extensively in animals. Characteristics of mutations in the C-terminus and the biological function of the new isoform, exon 1, were introduced. In studies on related neurodevelopmental disorders, a relationship is suggested between the MECP2 gene and autism-related gene, with overlapping pathways, but this is not common to other neurodevelopmental disorders. Developmental studies suggest an important role for MeCP2 in the formation and/or maintenance of synapses, and clarify the molecular biological aspects of Rett syndrome. However, early involvement of the aminergic neurons, suggested as the basic, pathognomonic lesion of Rett syndrome, has unfortunately not been investigated with the MECP2 mutation.

Genetic Factors in Catechol Estrogen Metabolism in Relation to the Risk of Endometrial Cancer

2-Hydroxylated metabolites of estrogen have been shown to have antiangiogenic effects and inhibit tumor cell proliferation, whereas 4-hydroxylated metabolites have been implicated in carcinogenesis. We examined whether polymorphisms in certain genes involved in estrogen metabolism are associated with endometrial cancer risk in a population-based case-control study with 371 cases and 420 controls. Based on previously published genotype-phenotype correlation studies, we defined variant alleles thought to increase estrogen 2-hydroxylation as presumptively low-risk (CYP1A1 m1 T6235C and m2 Ile(462)Val) and those thought to increase estrogen 4-hydroxylation as high-risk (CYP1A1 m4 Thr(461)Asn, CYP1A2 A734C, and CYP1B1 Leu(432)Val). Odds ratios (OR) and 95% confidence intervals (95% CI) were calculated using unconditional logistic regression. Carrying at least one CYP1A1 m1 or m2 variant allele was associated with a decreased risk of endometrial cancer [ORs (95% CIs), 0.64 (0.44-0.93) and 0.54 (0.30-0.99), respectively]. No strong alteration in risk was observed among women with any of the putative high-risk alleles. When CYP1A1, CYP1A2, and CYP1B1 genotypes were combined and ranked by the number of putative low-risk genotypes carried, women with four or five low-risk genotypes had a reduced risk of endometrial cancer (OR, 0.29; 95% CI, 0.15-0.56) compared with women with one or none. No appreciable alteration in risk was observed among women carrying two or three low-risk genotypes. Some of our findings are consistent with the hypothesis that increased estrogen 2-hydroxylation is associated with decreased endometrial cancer risk, but replication of these results is required before any firm conclusions can be reached.

A novel SCN1A mutation associated with severe GEFS+ in a large South American pedigree

Generalized epilepsy with febrile seizures plus (GEFS+) is an inherited epileptic syndrome with a marked clinical and genetic heterogeneity. Here we report the molecular characterization of a large pedigree with a severe clinical form of GEFS+. Genetic linkage analysis implied the involvement of the FEB3 in the disease phenotype of this family (parametric two-point lod-score of 2.2). Sequencing of the SCN1A gene revealed a novel aspartic acid for glycine substitution at position 1742 of this sodium channel subunit. The amino-acid replacement lies in the pore-forming region of domain IV of SCN1A. Our observations are consistent with the genotype-phenotype correlation studies suggesting that mutations in the pore-forming loop of SCN1A can lead to a clinically more severe epileptic syndrome.

Expression studies of mutations underlying Taiwanese Hunter syndrome (mucopolysaccharidosis type II)

Nearly 300 different mutations underlying mucopolysaccharidosis type II (MPS II) have been identified worldwide. To investigate the molecular lesions underlying Taiwanese MPS II, probands and families were identified and screened for iduronate-2-sulfatase (IDS) mutation by single-strand conformation polymorphism and DNA sequencing. Five novel and five previously reported mutations were found. Together with those previously reported, a total of 17 identified missense, small deletion, and nonsense mutations were further characterized by transient expression studies. Transfection of COS-7 cells by the mutated cDNA did not yield active enzyme, demonstrating the deleterious nature of the mutations. A 57% decrease in IDS mRNA level was seen with the 231del6 mutation. Among the 11 missense mutations examined, K347E substitution showed apparent normal maturation and targeting on immunoblot and confocal fluorescence microscopy examination. The other 10 missense mutations showed apparent normal precursor with little or reduced mature forms, indicating normal maturation but incorrect targeting of the mutant enzymes. Among the six deletion and nonsense mutations examined, 1055del12 and E521X showed abnormal maturation. The staining pattern of the truncated W267X and 1184delG proteins suggested retention within early vacuolar compartments. The mutated 231del6 and 1421delAG proteins were unstable and largely degraded. Molecular analysis of the IDS gene will clearly identify the cause of the disease within patients and allow antenatal and family studies. The further characterization of gene mutations may delineate their functional consequences on IDS activity and processing and may enable future studies of genotype-phenotype correlation to estimate a prognosis and to lead to possible therapeutic interventions.

Otolaryngologic markers for early diagnosis of turner syndrome

Problem: Turner syndrome (TS) affects up to one in 2,000 live female births and is characterized by the total or partial loss of one X chromosome in females with short stature and gonadal dysgenesis. Less penetrant features include sensorineural hearing loss (SNHL), otitis media (OM), pterygium colli, and craniofacial dysmorphism. We report the otolaryngologic features observed in a TS cohort. Methods: A cohort with a wide spectrum of phenotypic severity has been prospectively ascertained through a multidisciplinary study of genotype-phenotype correlation in TS at the National Institutes of Health. Subjects were evaluated by otolaryngologic examination, pure tone and speech audiometry, tympanometry, and stapedial reflex testing. Results: Ninety-nine TS patients (age range, 7 to 61 years; average, 30 ) with an average age at diagnosis of 10 years old (range, prenatal to 38 years) were studied. Sixty-three percent of the subjects had a high-arched or a peaked, ogival hard palate. Seventy-one percent had a history of recurrent OM and 64% had SNHL. Cross-sectional analysis revealed 2 predominant patterns of SNHL: adult-onset SNHL affecting high frequencies, and childhood-onset SNHL, initially affecting high or middle frequencies, with eventual progression to severe impairment across all frequencies. Conclusion: Diagnosis of TS is often delayed until the second decade or later. Almost all TS patients, even the least affected individuals with no external craniofacial stigmata of TS, had at least 1 of 3 otolaryngologic findings: recurrent OM, SNHL, and high-arched or ogival palate. Significance: The diagnosis of TS is often missed in female children presenting with OM and short stature. In association with a high-arched or ogival palate, or SNHL, these features are useful clinical markers for the initial detection and timely treatment of TS patients. Support: NIH intramural research funds Z01-DC-000064-03 and NICHD DIR Protocol 00-CH-0219.

Asymptomatic carriers for homozygous novel mutations in the FKRP gene: the other end of the spectrum.

Autosomal recessive limb-girdle muscular dystrophy linked to 19q13.3 (LGMD2I) was recently related to mutations in the fukutin-related protein gene (FKRP) gene. Pathogenic changes in the same gene were detected in congenital muscular dystrophy patients (MDC1C), a severe disorder. We have screened 86 LGMD genealogies to assess the frequency and distribution of mutations in the FKRP gene in Brazilian LGMD patients. We found 13 Brazilian genealogies, including 20 individuals with mutations in the FKRP gene, and identified nine novel pathogenic changes. The commonest C826A European mutation was found in 30% (9/26) of the mutated LGMD2I alleles. One affected patient homozygous for the FKRP (C826A) mutation also carries a missense R125H change in one allele of the caveolin-3 gene (responsible for LGMD1C muscular dystrophy). Two of her normal sibs were found to be double heterozygotes. In two unrelated LGMD2I families, homozygous for novel missense mutations, we identified four asymptomatic carriers, all older than 20 years. Genotype-phenotype correlation studies in the present study as well as in patients from different populations suggests that the spectrum of variability associated with mutations in the FKRP gene seems to be wider than in other forms of LGMD. It also reinforces the observations that pathogenic mutations are not always determinant of an abnormal phenotype, suggesting the possibility of other mechanisms modulating the severity of the phenotype that opens new avenues for therapeutic approaches.