Structural interpretation of mutations in phenylalanine hydroxylase protein aids in identifying genotype-phenotype correlations in phenylketonuria

Phenylalanine hydroxylase (PAH) is the enzyme that converts phenylalanine to tyrosine as a rate-limiting step in phenylalanine catabolism and protein and neurotransmitter biosynthesis. Over 300 mutations have been identified in the gene encoding PAH that result in a deficient enzyme activity and lead to the disorders hyperphenylalaninaemia and phenylketonuria. The determination of the crystal structure of PAH now allows the determination of the structural basis of mutations resulting in PAH deficiency. We present an analysis of the structural basis of 120 mutations with a 'classified' biochemical phenotype and/or available in vitro expression data. We find that the mutations can be grouped into five structural categories, based on the distinct expected structural and functional effects of the mutations in each category. Missense mutations and small amino acid deletions are found in three categories:'active site mutations', 'dimer interface mutations', and 'domain structure mutations'. Nonsense mutations and splicing mutations form the category of 'proteins with truncations and large deletions'. The final category, 'fusion proteins', is caused by frameshift mutations. We show that the structural information helps formulate some rules that will help predict the likely effects of unclassified and newly discovered mutations: proteins with truncations and large deletions, fusion proteins and active site mutations generally cause severe phenotypes; domain structure mutations and dimer interface mutations spread over a range of phenotypes, but domain structure mutations in the catalytic domain are more likely to be severe than domain structure mutations in the regulatory domain or dimer interface mutations.

Leber Congenital Amaurosis: Comprehensive Survey of the Genetic Heterogeneity, Refinement of the Clinical Definition, and Genotype-Phenotype Correlations as a Strategy for Molecular Diagnosis

Leber congenital amaurosis (LCA) is the earliest and most severe form of all inherited retinal dystrophies, responsible for congenital blindness. Disease-associated mutations have been hitherto reported in seven genes. These genes are all expressed preferentially in the photoreceptor cells or the retinal pigment epithelium but they are involved in strikingly different physiologic pathways resulting in an unforeseeable physiopathologic variety. This wide genetic and physiologic heterogeneity that could largely increase in the coming years, hinders the molecular diagnosis in LCA patients. The genotyping is, however, required to establish genetically defined subgroups of patients ready for therapy. Here, we report a comprehensive mutational analysis of the all known genes in 179 unrelated LCA patients, including 52 familial and 127 sporadic (27/127 consanguineous) cases. Mutations were identified in 47.5% patients. GUCY2D appeared to account for most LCA cases of our series (21.2%), followed by CRB1 (10%), RPE65 (6.1%), RPGRIP1 (4.5%), AIPL1 (3.4%), TULP1 (1.7%), and CRX (0.6%). The clinical history of all patients with mutations was carefully revisited to search for phenotype variations. Sound genotype-phenotype correlations were found that allowed us to divide patients into two main groups. The first one includes patients whose symptoms fit the traditional definition of LCA, i.e., congenital or very early cone-rod dystrophy, while the second group gathers patients affected with severe yet progressive rod-cone dystrophy. Besides, objective ophthalmologic data allowed us to subdivide each group into two subtypes. Based on these findings, we have drawn decisional flowcharts directing the molecular analysis of LCA genes in a given case. These flowcharts will hopefully lighten the heavy task of genotyping new patients but only if one has access to the most precise clinical history since birth.

Type I hyperprolinemia: genotype/phenotype correlations.

Type I hyperprolinemia (HPI) is an autosomal recessive disorder associated with cognitive and psychiatric troubles, caused by alterations of the Proline Dehydrogenase gene (PRODH) at 22q11. HPI results from PRODH deletion and/or missense mutations reducing proline oxidase (POX) activity. The goals of this study were first to measure in controls the frequency of PRODH variations described in HPI patients, second to assess the functional effect of PRODH mutations on POX activity, and finally to establish genotype/enzymatic activity correlations in a new series of HPI patients. Eight of 14 variants occurred at polymorphic frequency in 114 controls. POX activity was determined for six novel mutations and two haplotypes. The c.1331G>A, p.G444D allele has a drastic effect, whereas the c.23C>T, p.P8L allele and the c.[56C>A; 172G>A], p.[Q19P; A58T] haplotype result in a moderate decrease in activity. Among the 19 HPI patients, 10 had a predicted residual activity <50%. Eight out of nine subjects with a predicted residual activity > or = 50% bore at least one c.824C>A, p.T275N allele, which has no detrimental effect on activity but whose frequency in controls is only 3%. Our results suggest that PRODH mutations lead to a decreased POX activity or affect other biological parameters causing hyperprolinemia.

Complement Factor B Polymorphism and the Phenotype of Early Age-related Macular Degeneration.

Abstract Purpose: Age-related macular degeneration (AMD) has been associated with a number of polymorphisms in genes in the complement pathway. We examined the potential genotype-phenotype correlation of complement factor B (CFB) (R32Q) polymorphisms in Caucasian patients with AMD. Methods: Data from a Central European cohort of 349 patients with early AMD in at least one eye were analyzed for potential associations of the CFB (R32Q/rs641153) polymorphism with phenotypic features of early AMD. Early AMD was classified according to the International Classification and Grading System into predominant drusen size, largest drusen, drusen covered surface, central or ring-like location, peripheral drusen, and pigmentary changes. The potential association with single nucleotide polymorphisms on CFB (R32Q/rs641153) was evaluated for all patients, corrected for age, sex, and the polymorphisms of CFH (Y402H) and ARMS2 (A69S). Results: CFB (R32Q) polymorphisms showed a significant association with smaller drusen size (largest drusen ≤250 µm, p = 0.021, predominant drusen ≤125 µm, p = 0.016), with smaller surface covered by drusen (≤10%; p = 0.02), and with more frequent occurrence of peripheral drusen (p = 0.007). No association was found for pigmentary changes. Conclusions: The CFB (R32Q) polymorphism was associated with AMD characterized by small drusen only, and appeared to be protective of large drusen (OR 0.48/0.45) and of larger drusen covered area (OR 0.34). Furthermore, peripheral drusen were more frequently found (OR 2.27). This result supports the role of complement components and their polymorphisms in drusen formation and may enable a better understanding of AMD pathogenesis.

Identification of an RP1 Prevalent Founder Mutation and Related Phenotype in Spanish Patients with Early-Onset Autosomal Recessive Retinitis.

OBJECTIVE: To identify the genetic causes underlying early-onset autosomal recessive retinitis pigmentosa (arRP) in the Spanish population and describe the associated phenotype. DESIGN: Case series. PARTICIPANTS: A total of 244 unrelated families affected by early-onset arRP. METHODS: Homozygosity mapping or exome sequencing analysis was performed in 3 families segregating arRP. A mutational screening was performed in 241 additional unrelated families for the p.Ser452Stop mutation. Haplotype analysis also was conducted. Individuals who were homozygotes, double heterozygotes, or carriers of mutations in RP1 underwent an ophthalmic evaluation to establish a genotype-phenotype correlation. MAIN OUTCOME MEASURES: DNA sequence variants, homozygous regions, haplotypes, best-corrected visual acuity, visual field assessments, electroretinogram responses, and optical coherence tomography images. RESULTS: Four novel mutations in RP1 were identified. The new mutation p.Ser542Stop was present in 11 of 244 (4.5%) of the studied families. All chromosomes harboring this mutation shared the same haplotype. All patients presented a common phenotype with an early age of onset and a prompt macular degeneration, whereas the heterozygote carriers did not show any signs of retinitis pigmentosa (RP). CONCLUSIONS: p.Ser542Stop is a single founder mutation and the most prevalent described mutation in the Spanish population. It causes early-onset RP with a rapid macular degeneration and is responsible for 4.5% of all cases. Our data suggest that the implication of RP1 in arRP may be underestimated. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Genotype-phenotype analysis of Jervell and Lange-Nielsen syndrome in six families from Saudi Arabia.

We sought to explore the genotype-phenotype of Jervell and Lange-Nielsen syndrome (JLNS) patients in Saudi Arabia. We have also assessed the plausible effect of consanguinity into the pathology of JLNS. Six families with at least one JLNS-affected member attended our clinic between 2011 and 2013. Retrospective and prospective clinical data were collected and genetic investigation was performed. Pathogenic mutations in the KCNQ1 gene were detected in all JLNS patients. The homozygous mutations detected were Leu273Phe, Asp202Asn, Ile567Thr, and c.1486_1487delCT and compound heterozygous mutations were c.820_ 830del and c.1251+1G>T. All living JLNS patients except one had a QTc of >500 ms and a history of recurrent syncope. β-Blockers abolished the cardiac-related events in all patients except two siblings with homozygous Ile567Thr mutation. Four of the six mutations were originally reported in autosomal dominant long QT syndrome (LQTS) patients. Eighty percent of the heterozygote mutation carriers showed prolongation of QTc, but majority of these reported no symptoms attributable to arrhythmias. Mutations detected in this study will be advantageous in tribe and region-specific cascade screening of LQTS in Saudi Arabia.

Para-arterial sparing phenotype in Leber congenital amaurosis associated with homozygous p.R108X SPATA-7 mutation

Purpose: To describe the genotype/phenotype correlation associated with homozygous p.R108X mutation in the SPATA7 gene. Methods: A consanguineous nuclear family of Ethiopian origin was ascertained for genotypic and phenotypic characterization, including fundus photography, fundus autofluorescence (FAF), and full-field ERG. Molecular diagnosis was performed using a microarray. Results: Two of the 5 family members were affected with LCA. A homozygous c.322C>T (p.R108X) mutation in exon 5 of SPATA-7 was identified in both of them. The patients were 4 and 11 years old, respectively. Fundus examination revealed an unremarkable macular area, but optic nerve pallor, attenuated vascular calibre and deep retinal nummular deposits with para-arterial sparing predominant in the midperiphery. FAF showed multiples areas of hyperautofluorescence, corresponding to the deep retinal deposits. ERG was not recordable in the young patient, and showed severe rods/cones dysfunction in the older one. Conclusions: The literature describing genotype/phenotype correlation of SPATA-7 mutations in Leber congenital amaurosis (LCA) is still limited. We report the occurrence of para-arterial sparing in two sibs with SPATA7-linked LCA which may represent a clinical marker of this condition.

An overview of L-2-hydroxyglutarate dehydrogenase gene (L2HGDH) variants: a genotype-phenotype study.

L-2-Hydroxyglutaric aciduria (L2HGA) is a rare, neurometabolic disorder with an autosomal recessive mode of inheritance. Affected individuals only have neurological manifestations, including psychomotor retardation, cerebellar ataxia, and more variably macrocephaly, or epilepsy. The diagnosis of L2HGA can be made based on magnetic resonance imaging (MRI), biochemical analysis, and mutational analysis of L2HGDH. About 200 patients with elevated concentrations of 2-hydroxyglutarate (2HG) in the urine were referred for chiral determination of 2HG and L2HGDH mutational analysis. All patients with increased L2HG (n=106; 83 families) were included. Clinical information on 61 patients was obtained via questionnaires. In 82 families the mutations were detected by direct sequence analysis and/or multiplex ligation dependent probe amplification (MLPA), including one case where MLPA was essential to detect the second allele. In another case RT-PCR followed by deep intronic sequencing was needed to detect the mutation. Thirty-five novel mutations as well as 35 reported mutations and 14 nondisease-related variants are reviewed and included in a novel Leiden Open source Variation Database (LOVD) for L2HGDH variants (http://www.LOVD.nl/L2HGDH). Every user can access the database and submit variants/patients. Furthermore, we report on the phenotype, including neurological manifestations and urinary levels of L2HG, and we evaluate the phenotype-genotype relationship.

Thr(118)Met amino acid substitution in the peripheral myelin protein 22 does not influence the clinical phenotype of Charcot-Marie-Tooth disease type 1A due to the 17p11.2-p12 duplication

The Thr(118)Met substitution in the peripheral myelin protein 22 (PMP22) gene has been detected in a number of families with demyelinating Charcot-Marie-Tooth (CMT1) neuropathy or with the hereditary neuropathy with liability to pressure palsy, but in none of them has it consistently segregated with the peripheral neuropathy. We describe here a CMT1 family (a 63-year-old man, his brother and his niece) in which two mutations on different chromosomes were found in the PMP22 gene, the 17p duplication, detected by fluorescent semiquantitative polymerase chain reaction (PCR) of microsatellite markers localized within the duplicated region on chromosome 17p11.2-p12, and the Thr(118)Met substitution, detected by direct sequencing the four coding exons of the PMP22 gene. A genotype/phenotype correlation study showed that the neuropathy segregates with the duplication and that the amino acid substitution does not seem to modify the clinical characteristics or the severity of the peripheral neuropathy. We did not find any evidence to characterize this substitution as a polymorphism in the population studied and we propose that the high frequency reported for this point mutation in the literature suggests that the Thr(118)Met substitution may be a hotspot for mutations in the PMP22 gene.

On the traces of XPD: cell cycle matters - untangling the genotype-phenotype relationship of XPD mutations

Abstract Mutations in the human gene coding for XPD lead to segmental progeria - the premature appearance of some of the phenotypes normally associated with aging - which may or may not be accompanied by increased cancer incidence. XPD is required for at least three different critical cellular functions: in addition to participating in the process of nucleotide excision repair (NER), which removes bulky DNA lesions, XPD also regulates transcription as part of the general transcription factor IIH (TFIIH) and controls cell cycle progression through its interaction with CAK, a pivotal activator of cyclin dependent kinases (CDKs). The study of inherited XPD disorders offers the opportunity to gain insights into the coordination of important cellular events and may shed light on the mechanisms that regulate the delicate equilibrium between cell proliferation and functional senescence, which is notably altered during physiological aging and in cancer. The phenotypic manifestations in the different XPD disorders are the sum of disturbances in the vital processes carried out by TFIIH and CAK. In addition, further TFIIH- and CAK-independent cellular activities of XPD may also play a role. This, added to the complex feedback networks that are in place to guarantee the coordination between cell cycle, DNA repair and transcription, complicates the interpretation of clinical observations. While results obtained from patient cell isolates as well as from murine models have been elementary in revealing such complexity, the Drosophila embryo has proven useful to analyze the role of XPD as a cell cycle regulator independently from its other cellular functions. Together with data from the biochemical and structural analysis of XPD and of the TFIIH complex these results combine into a new picture of the XPD activities that provides ground for a better understanding of the patophysiology of XPD diseases and for future development of diagnostic and therapeutic tools.

Single nucleotide polymorphism (SNP) selection for genotype-phenotype association studies

With hundreds of single nucleotide polymorphisms (SNPs) in a candidate gene and millions of SNPs across the genome, selecting an informative subset of SNPs to maximize the ability to detect genotype-phenotype association is of great interest and importance. In addition, with a large number of SNPs, analytic methods are needed that allow investigators to control the false positive rate resulting from large numbers of SNP genotype-phenotype analyses. This dissertation uses simulated data to explore methods for selecting SNPs for genotype-phenotype association studies. I examined the pattern of linkage disequilibrium (LD) across a candidate gene region and used this pattern to aid in localizing a disease-influencing mutation. The results indicate that the r2 measure of linkage disequilibrium is preferred over the common D′ measure for use in genotype-phenotype association studies. Using step-wise linear regression, the best predictor of the quantitative trait was not usually the single functional mutation. Rather it was a SNP that was in high linkage disequilibrium with the functional mutation. Next, I compared three strategies for selecting SNPs for application to phenotype association studies: based on measures of linkage disequilibrium, based on a measure of haplotype diversity, and random selection. The results demonstrate that SNPs selected based on maximum haplotype diversity are more informative and yield higher power than randomly selected SNPs or SNPs selected based on low pair-wise LD. The data also indicate that for genes with small contribution to the phenotype, it is more prudent for investigators to increase their sample size than to continuously increase the number of SNPs in order to improve statistical power. When typing large numbers of SNPs, researchers are faced with the challenge of utilizing an appropriate statistical method that controls the type I error rate while maintaining adequate power. We show that an empirical genotype based multi-locus global test that uses permutation testing to investigate the null distribution of the maximum test statistic maintains a desired overall type I error rate while not overly sacrificing statistical power. The results also show that when the penetrance model is simple the multi-locus global test does as well or better than the haplotype analysis. However, for more complex models, haplotype analyses offer advantages. The results of this dissertation will be of utility to human geneticists designing large-scale multi-locus genotype-phenotype association studies. ^

Functional data analysis approaches for genotype-phenotype association studies from next-generation sequencing

Next-generation DNA sequencing platforms can effectively detect the entire spectrum of genomic variation and is emerging to be a major tool for systematic exploration of the universe of variants and interactions in the entire genome. However, the data produced by next-generation sequencing technologies will suffer from three basic problems: sequence errors, assembly errors, and missing data. Current statistical methods for genetic analysis are well suited for detecting the association of common variants, but are less suitable to rare variants. This raises great challenge for sequence-based genetic studies of complex diseases.^ This research dissertation utilized genome continuum model as a general principle, and stochastic calculus and functional data analysis as tools for developing novel and powerful statistical methods for next generation of association studies of both qualitative and quantitative traits in the context of sequencing data, which finally lead to shifting the paradigm of association analysis from the current locus-by-locus analysis to collectively analyzing genome regions.^ In this project, the functional principal component (FPC) methods coupled with high-dimensional data reduction techniques will be used to develop novel and powerful methods for testing the associations of the entire spectrum of genetic variation within a segment of genome or a gene regardless of whether the variants are common or rare.^ The classical quantitative genetics suffer from high type I error rates and low power for rare variants. To overcome these limitations for resequencing data, this project used functional linear models with scalar response to develop statistics for identifying quantitative trait loci (QTLs) for both common and rare variants. To illustrate their applications, the functional linear models were applied to five quantitative traits in Framingham heart studies. ^ This project proposed a novel concept of gene-gene co-association in which a gene or a genomic region is taken as a unit of association analysis and used stochastic calculus to develop a unified framework for testing the association of multiple genes or genomic regions for both common and rare alleles. The proposed methods were applied to gene-gene co-association analysis of psoriasis in two independent GWAS datasets which led to discovery of networks significantly associated with psoriasis.^

DETERMINING THE GENOTYPE-PHENOTYPE CONNECTION IN SYNTHETIC INDUCIBLE GENE EXPRESSION SYSTEMS

Introduction Gene expression is an important process whereby the genotype controls an individual cell’s phenotype. However, even genetically identical cells display a variety of phenotypes, which may be attributed to differences in their environment. Yet, even after controlling for these two factors, individual phenotypes still diverge due to noisy gene expression. Synthetic gene expression systems allow investigators to isolate, control, and measure the effects of noise on cell phenotypes. I used mathematical and computational methods to design, study, and predict the behavior of synthetic gene expression systems in S. cerevisiae, which were affected by noise. Methods I created probabilistic biochemical reaction models from known behaviors of the tetR and rtTA genes, gene products, and their gene architectures. I then simplified these models to account for essential behaviors of gene expression systems. Finally, I used these models to predict behaviors of modified gene expression systems, which were experimentally verified. Results Cell growth, which is often ignored when formulating chemical kinetics models, was essential for understanding gene expression behavior. Models incorporating growth effects were used to explain unexpected reductions in gene expression noise, design a set of gene expression systems with “linear” dose-responses, and quantify the speed with which cells explored their fitness landscapes due to noisy gene expression. Conclusions Models incorporating noisy gene expression and cell division were necessary to design, understand, and predict the behaviors of synthetic gene expression systems. The methods and models developed here will allow investigators to more efficiently design new gene expression systems, and infer gene expression properties of TetR based systems.