Long-read genome sequencing identifies cryptic structural variants in congenital aniridia cases.

BACKGROUND: Haploinsufficiency of the transcription factor PAX6 is the main cause of congenital aniridia, a genetic disorder characterized by iris and foveal hypoplasia. 11p13 microdeletions altering PAX6 or its downstream regulatory region (DRR) are present in about 25% of patients; however, only a few complex rearrangements have been described to date. Here, we performed nanopore-based whole-genome sequencing to assess the presence of cryptic structural variants (SVs) on the only two unsolved "PAX6-negative" cases from a cohort of 110 patients with congenital aniridia after unsuccessfully short-read sequencing approaches. RESULTS: Long-read sequencing (LRS) unveiled balanced chromosomal rearrangements affecting the PAX6 locus at 11p13 in these two patients and allowed nucleotide-level breakpoint analysis. First, we identified a cryptic 4.9 Mb de novo inversion disrupting intron 7 of PAX6, further verified by targeted polymerase chain reaction amplification and sequencing and FISH-based cytogenetic analysis. Furthermore, LRS was decisive in correctly mapping a t(6;11) balanced translocation cytogenetically detected in a second proband with congenital aniridia and considered non-causal 15 years ago. LRS resolved that the breakpoint on chromosome 11 was indeed located at 11p13, disrupting the DNase I hypersensitive site 2 enhancer within the DRR of PAX6, 161 Kb from the causal gene. Patient-derived RNA expression analysis demonstrated PAX6 haploinsufficiency, thus supporting that the 11p13 breakpoint led to a positional effect by cleaving crucial enhancers for PAX6 transactivation. LRS analysis was also critical for mapping the exact breakpoint on chromosome 6 to the highly repetitive centromeric region at 6p11.1. CONCLUSIONS: In both cases, the LRS-based identified SVs have been deemed the hidden pathogenic cause of congenital aniridia. Our study underscores the limitations of traditional short-read sequencing in uncovering pathogenic SVs affecting low-complexity regions of the genome and the value of LRS in providing insight into hidden sources of variation in rare genetic diseases.

Clinical spectrum of MTOR-related hypomelanosis of Ito with neurodevelopmental abnormalities.

PURPOSE: Hypomelanosis of Ito (HI) is a skin marker of somatic mosaicism. Mosaic MTOR pathogenic variants have been reported in HI with brain overgrowth. We sought to delineate further the pigmentary skin phenotype and clinical spectrum of neurodevelopmental manifestations of MTOR-related HI. METHODS: From two cohorts totaling 71 patients with pigmentary mosaicism, we identified 14 patients with Blaschko-linear and one with flag-like pigmentation abnormalities, psychomotor impairment or seizures, and a postzygotic MTOR variant in skin. Patient records, including brain magnetic resonance image (MRI) were reviewed. Immunostaining (n = 3) for melanocyte markers and ultrastructural studies (n = 2) were performed on skin biopsies. RESULTS: MTOR variants were present in skin, but absent from blood in half of cases. In a patient (p.[Glu2419Lys] variant), phosphorylation of p70S6K was constitutively increased. In hypopigmented skin of two patients, we found a decrease in stage 4 melanosomes in melanocytes and keratinocytes. Most patients (80%) had macrocephaly or (hemi)megalencephaly on MRI. CONCLUSION: MTOR-related HI is a recognizable neurocutaneous phenotype of patterned dyspigmentation, epilepsy, intellectual deficiency, and brain overgrowth, and a distinct subtype of hypomelanosis related to somatic mosaicism. Hypopigmentation may be due to a defect in melanogenesis, through mTORC1 activation, similar to hypochromic patches in tuberous sclerosis complex.

Meta-analysis of SHANK Mutations in Autism Spectrum Disorders: a gradient of severity in cognitive impairments.

SHANK genes code for scaffold proteins located at the post-synaptic density of glutamatergic synapses. In neurons, SHANK2 and SHANK3 have a positive effect on the induction and maturation of dendritic spines, whereas SHANK1 induces the enlargement of spine heads. Mutations in SHANK genes have been associated with autism spectrum disorders (ASD), but their prevalence and clinical relevance remain to be determined. Here, we performed a new screen and a meta-analysis of SHANK copy-number and coding-sequence variants in ASD. Copy-number variants were analyzed in 5,657 patients and 19,163 controls, coding-sequence variants were ascertained in 760 to 2,147 patients and 492 to 1,090 controls (depending on the gene), and, individuals carrying de novo or truncating SHANK mutations underwent an extensive clinical investigation. Copy-number variants and truncating mutations in SHANK genes were present in ∼1% of patients with ASD: mutations in SHANK1 were rare (0.04%) and present in males with normal IQ and autism; mutations in SHANK2 were present in 0.17% of patients with ASD and mild intellectual disability; mutations in SHANK3 were present in 0.69% of patients with ASD and up to 2.12% of the cases with moderate to profound intellectual disability. In summary, mutations of the SHANK genes were detected in the whole spectrum of autism with a gradient of severity in cognitive impairment. Given the rare frequency of SHANK1 and SHANK2 deleterious mutations, the clinical relevance of these genes remains to be ascertained. In contrast, the frequency and the penetrance of SHANK3 mutations in individuals with ASD and intellectual disability-more than 1 in 50-warrant its consideration for mutation screening in clinical practice.

Mutations in BCAP31 cause a severe X-linked phenotype with deafness, dystonia, and central hypomyelination and disorganize the Golgi apparatus.

BAP31 is one of the most abundant endoplasmic reticulum (ER) membrane proteins. It is a chaperone protein involved in several pathways, including ER-associated degradation, export of ER proteins to the Golgi apparatus, and programmed cell death. BAP31 is encoded by BCAP31, located in human Xq28 and highly expressed in neurons. We identified loss-of-function mutations in BCAP31 in seven individuals from three families. These persons suffered from motor and intellectual disabilities, dystonia, sensorineural deafness, and white-matter changes, which together define an X-linked syndrome. In the primary fibroblasts of affected individuals, we found that BCAP31 deficiency altered ER morphology and caused a disorganization of the Golgi apparatus in a significant proportion of cells. Contrary to what has been described with transient-RNA-interference experiments, we demonstrate that constitutive BCAP31 deficiency does not activate the unfolded protein response or cell-death effectors. Rather, our data demonstrate that the lack of BAP31 disturbs ER metabolism and impacts the Golgi apparatus, highlighting an important role for BAP31 in ER-to-Golgi crosstalk. These findings provide a molecular basis for a Mendelian syndrome and link intracellular protein trafficking to severe congenital brain dysfunction and deafness.

Genetic and functional analyses of SHANK2 mutations suggest a multiple hit model of autism spectrum disorders.

Autism spectrum disorders (ASD) are a heterogeneous group of neurodevelopmental disorders with a complex inheritance pattern. While many rare variants in synaptic proteins have been identified in patients with ASD, little is known about their effects at the synapse and their interactions with other genetic variations. Here, following the discovery of two de novo SHANK2 deletions by the Autism Genome Project, we identified a novel 421 kb de novo SHANK2 deletion in a patient with autism. We then sequenced SHANK2 in 455 patients with ASD and 431 controls and integrated these results with those reported by Berkel et al. 2010 (n = 396 patients and n = 659 controls). We observed a significant enrichment of variants affecting conserved amino acids in 29 of 851 (3.4%) patients and in 16 of 1,090 (1.5%) controls (P = 0.004, OR = 2.37, 95% CI = 1.23-4.70). In neuronal cell cultures, the variants identified in patients were associated with a reduced synaptic density at dendrites compared to the variants only detected in controls (P = 0.0013). Interestingly, the three patients with de novo SHANK2 deletions also carried inherited CNVs at 15q11-q13 previously associated with neuropsychiatric disorders. In two cases, the nicotinic receptor CHRNA7 was duplicated and in one case the synaptic translation repressor CYFIP1 was deleted. These results strengthen the role of synaptic gene dysfunction in ASD but also highlight the presence of putative modifier genes, which is in keeping with the "multiple hit model" for ASD. A better knowledge of these genetic interactions will be necessary to understand the complex inheritance pattern of ASD.

Perilipin deficiency and autosomal dominant partial lipodystrophy.

Perilipin is the most abundant adipocyte-specific protein that coats lipid droplets, and it is required for optimal lipid incorporation and release from the droplet. We identified two heterozygous frameshift mutations in the perilipin gene (PLIN1) in three families with partial lipodystrophy, severe dyslipidemia, and insulin-resistant diabetes. Subcutaneous fat from the patients was characterized by smaller-than-normal adipocytes, macrophage infiltration, and fibrosis. In contrast to wild-type perilipin, mutant forms of the protein failed to increase triglyceride accumulation when expressed heterologously in preadipocytes. These findings define a novel dominant form of inherited lipodystrophy and highlight the serious metabolic consequences of a primary defect in the formation of lipid droplets in adipose tissue.

WFS1 mutations are frequent monogenic causes of juvenile-onset diabetes mellitus in Lebanon.

Most cases of juvenile-onset diabetes (JOD) are diagnosed as type 1 diabetes (T1D), for which genetic studies conducted in outbred Caucasian populations support the concept of multifactorial inheritance. However, this view may be partly challenged in particular population settings. In view of the suggestive evidence for a high prevalence of Wolfram syndrome (WFS) in Lebanon, the phenotypic variability associated with WFS1 mutations, and the high consanguinity rate in Lebanon, we aimed to evaluate the contribution of WFS1 mutations as monogenic determinants to JOD in Lebanon. We performed a family-based genetic study, with linkage analysis followed by systematic mutation screening of WFS1 exons in all JOD probands. The study population consisted of an unbiased recruitment of all juvenile-onset insulin-dependent diabetic patients from a specialized diabetes pediatric clinic in Beirut, Lebanon. Homozygous or compound heterozygous WFS1 mutations were found in 22 of the 399 JOD probands (5.5%), resulting in WFS (17 probands) or in non-syndromic non-autoimmune diabetes mellitus (DM, five probands). These accounted for 12.1% (21/174) of probands in consanguineous families, compared with 0.4% (1/225) in non-consanguineous families. Of the 38 patients identified with homozygous or compound heterozygous WFS1 mutations, 11 (29%) had non-syndromic DM, all of whom carried a particular WFS1 mutation, WFS1(LIB), encoding a protein with an extended C-terminal domain. This mutation resulted in a delayed onset or absence of extrapancreatic features. These results underscore the major impact of population-specific factors, such as population-specific mutations and founder effects, and family structure in the genetic determinism of JOD.

Genome sequencing in cytogenetics: Comparison of short-read and linked-read approaches for germline structural variant detection and characterization.

BACKGROUND: Structural variants (SVs) include copy number variants (CNVs) and apparently balanced chromosomal rearrangements (ABCRs). Genome sequencing (GS) enables SV detection at base-pair resolution, but the use of short-read sequencing is limited by repetitive sequences, and long-read approaches are not yet validated for diagnosis. Recently, 10X Genomics proposed Chromium, a technology providing linked-reads to reconstruct long DNA fragments and which could represent a good alternative. No study has compared short-read to linked-read technologies to detect SVs in a constitutional diagnostic setting yet. The aim of this work was to determine whether the 10X Genomics technology enables better detection and comprehension of SVs than short-read WGS. METHODS: We included 13 patients carrying various SVs. Whole genome analyses were performed using paired-end HiSeq X sequencing with (linked-read strategy) or without (short-read strategy) Chromium library preparation. Two different bioinformatic pipelines were used: Variants are called using BreakDancer for short-read strategy and LongRanger for long-read strategy. Variant interpretations were first blinded. RESULTS: The short-read strategy allowed diagnosis of known SV in 10/13 patients. After unblinding, the linked-read strategy identified 10/13 SVs, including one (patient 7) missed by the short-read strategy. CONCLUSION: In conclusion, regarding the results of this study, 10X Genomics solution did not improve the detection and characterization of SV.

Association of a homozygous nonsense caveolin-1 mutation with Berardinelli-Seip congenital lipodystrophy.

CONTEXT: Berardinelli-Seip congenital lipodystrophy (BSCL) is a rare recessive disease characterized by near absence of adipose tissue, resulting in severe dyslipidemia and insulin resistance. In most reported cases, BSCL is due to alterations in either seipin, of unknown function, or 1-acylglycerol-3-phosphate acyltransferase-beta (AGPAT2), which catalyzes the formation of phosphatidic acid. OBJECTIVE: We sought to determine the genetic origin of the unexplained cases of BSCL. We thus sequenced CAV1, encoding caveolin-1, as a candidate gene involved in insulin signaling and lipid homeostasis. CAV1 is a key structural component of plasma membrane caveolae, and Cav1-deficient mice display progressive loss of adipose tissue and insulin resistance. DESIGN: We undertook phenotyping studies and molecular screening of CAV1 in four patients with BSCL with no mutation in the genes encoding either seipin or AGPAT2. RESULTS: A homozygous nonsense mutation (p.Glu38X) was identified in CAV1 in a patient with BSCL born from a consanguineous union. This mutation affects both the alpha- and beta-CAV1 isoforms and ablates CAV1 expression in skin fibroblasts. Detailed magnetic resonance imaging of the proband confirmed near total absence of both sc and visceral adipose tissue, with only vestigial amounts in the dorsal sc regions. In keeping with the lack of adipose tissue, the proband was also severely insulin resistant and dyslipidemic. In addition, the proband had mild hypocalcemia likely due to vitamin D resistance. CONCLUSIONS: These findings identify CAV1 as a new BSCL-related gene and support a critical role for caveolins in human adipocyte function.

Performance comparison of three DNA extraction kits on human whole-exome data from formalin-fixed paraffin-embedded normal and tumor samples.

Next-generation sequencing (NGS) studies are becoming routinely used for the detection of novel and clinically actionable DNA variants at a pangenomic scale. Such analyses are now used in the clinical practice to enable precision medicine. Formalin-fixed paraffin-embedded (FFPE) tissues are still one of the most abundant source of cancer clinical specimen, unfortunately this method of preparation is known to degrade DNA and therefore compromise subsequent analysis. Some studies have reported that variant detection can be performed on FFPE samples sequenced with NGS techniques, but few or none have done an in-depth coverage analysis and compared the influence of different state-of-the-art FFPE DNA extraction kits on the quality of the variant calling. Here, we generated 42 human whole-exome sequencing data sets from fresh-frozen (FF) and FFPE samples. These samples include normal and tumor tissues from two different organs (liver and colon), that we extracted with three different FFPE extraction kits (QIAamp DNA FFPE Tissue kit and GeneRead DNA FFPE kit from Qiagen, Maxwell™ RSC DNA FFPE Kit from Promega). We determined the rate of concordance of called variants between matched FF and FFPE samples on all common variants (representing at least 86% of the total number of variants for SNVs). The concordance rate is very high between all matched FF / FFPE pairs, with equivalent values for the three kits we analyzed. On the other hand, when looking at the difference between the total number of variants in FF and FFPE, we find a significant variation for the three different FFPE DNA extraction kits. Coverage analysis shows that FFPE samples have less good indicators than FF samples, yet the coverage quality remains above accepted thresholds. We detect limited but statistically significant variations in coverage indicator values between the three FFPE extraction kits. Globally, the GeneRead and QIAamp kits have better variant calling and coverage indicators than the Maxwell kit on the samples used in this study, although this kit performs better on some indicators and has advantages in terms of practical usage. Taken together, our results confirm the potential of FFPE samples analysis for clinical genomic studies, but also indicate that the choice of a FFPE DNA extraction kit should be done with careful testing and analysis beforehand in order to maximize the accuracy of the results.

Fibrinogen and coronary heart disease: test of causality by 'Mendelian randomization'.

BACKGROUND: Blood concentrations of fibrinogen have been associated with coronary heart disease risk in epidemiological studies, but it is uncertain whether this association is causal or reflects residual confounding by other risk factors. We investigated the relationship between the single nucleotide polymorphism at position -148 in the beta-fibrinogen gene promoter (beta - 148C/T), blood fibrinogen levels, and risk of myocardial infarction (MI) in sufficiently large numbers of coronary disease cases to reliably address this question. METHODS: Genotyping and measurement of blood fibrinogen concentration were carried out in 4,685 cases of confirmed MI and 3,460 controls with no history of coronary disease. A meta-analysis of ISIS and 19 other studies of beta-fibrinogen genotypes involving a total of 12,220 coronary disease cases and 18,716 controls was conducted. RESULTS: Among the ISIS controls, mean plasma fibrinogen concentrations with the C/C, C/T and T/T genotypes were 3.34 (SE 0.015), 3.48 (0.022), and 3.60 (0.064) g/l, respectively, corresponding to an increase of 0.14 (0.024) g/l per T allele (trend P < 0.0001). In the case-control comparison, 0.14 g/l higher usual plasma fibrinogen concentration was associated with an age-adjusted and sex-adjusted risk ratio for MI of 1.17 [95% confidence interval (95% CI) 1.14-1.19; P < 0.0001]. But, after further adjustment for smoking, body mass index, and plasma apolipoprotein B/A(1) ratio, this risk ratio fell to 1.03 (95% CI 1.00-1.05; P = 0.05). Moreover, fibrinogen genotype was not significantly associated with MI incidence: risk ratio of 1.06 (95% CI 0.96-1.16) per higher-fibrinogen allele in ISIS alone and of 1.00 (95% CI 0.95-1.04) per allele in the meta-analysis. CONCLUSIONS: Genotypes that produce lifelong differences in fibrinogen concentrations do not materially influence coronary disease incidence. As these genotype-dependent differences in fibrinogen were allocated randomly at conception (Mendelian randomization), this association is not likely to be confounded by other factors. Consequently, these genetic results provide strong evidence that long-term differences in fibrinogen concentrations are not a major determinant of coronary disease risk.

Maladaptative Autophagy Impairs Adipose Function in Congenital Generalized Lipodystrophy due to Cavin-1 Deficiency.

CONTEXT: Mutations in PTRF encoding cavin-1 are responsible for congenital generalized lipodystrophy type 4 (CGL4) characterized by lipoatrophy, insulin resistance, dyslipidemia, and muscular dystrophy. Cavin-1 cooperates with caveolins to form the plasma membrane caveolae, which are involved in cellular trafficking and signalling and in lipid turnover. OBJECTIVE: We sought to identify PTRF mutations in patients with CGL and to determine their impact on insulin sensitivity, adipose differentiation, and cellular autophagy. DESIGN AND PATIENTS: We performed phenotyping studies and molecular screening of PTRF in two unrelated families with CGL. Cellular studies were conducted in cultured skin fibroblasts from the two probands and from control subjects, and in murine 3T3-F442A preadipocytes. Knockdown of cavin-1 or ATG5 was obtained by small interfering RNA-mediated silencing. RESULTS: We identified two new PTRF homozygous mutations (p.Asp59Val or p.Gln157Hisfs*52) in four patients with CGL4 presenting with generalized lipoatrophy and associated metabolic abnormalities. In probands' fibroblasts, cavin-1 expression was undetectable and caveolin-1 and -2 barely expressed. Ultrastructural analysis revealed a loss of membrane caveolae and the presence of numerous cytoplasmic autophagosomes. Patients' cells also showed increased autophagic flux and blunted insulin signaling. These results were reproduced by PTRF knockdown in control fibroblasts and in 3T3-F442A preadipocytes. Cavin-1 deficiency also impaired 3T3-F442A adipocyte differentiation. Suppression of autophagy by small interfering RNA-mediated silencing of ATG5 improved insulin sensitivity and adipocyte differentiation. CONCLUSIONS: This study showed that cavin-1 deficiency resulted in maladaptative autophagy that contributed to insulin resistance and altered adipocyte differentiation. These new pathophysiological mechanisms could open new therapeutic perspectives for adipose tissue diseases including CGL4.

Variations of SOST mRNA expression in human bone are associated with DNA polymorphism and DNA methylation in the SOST gene.

SOST encodes sclerostin, an inhibitor of bone formation that antagonizes canonical Wnt signaling. Variations of SOST expression have an impact on bone mineral density (BMD) and bone strength. We hypothesized that genetic and epigenetic DNA modifications have an impact on SOST gene expression. By analyzing 43 bone samples from women, we found that rs851054 (G/A) is associated with SOST mRNA expression, donors with one or two G allele(s) displaying higher SOST expression (p<0.05). Beside this polymorphism, we also investigated the role of CpG methylation in SOST mRNA expression, and analyzed methylation variation at 13 CpG sites on the 1st exon of SOST in 14 human bone samples. Principal component analysis identified three groups of CpG sites that explained most of the methylation variation. We calculated the percentage of methylation in the main group of CpGs, and showed that higher rates of methylated CpGs are associated with higher SOST mRNA expression. To demonstrate that change in SOST expression might be related to human bone disease, we analyzed 131 patients with osteogenesis imperfecta (OI), a rare disease characterized by low BMD, bone fragility, and marked intra-familial variability of bone phenotypes. We found an association between rs851054 of the SOST promoter and the fracture rate only during childhood (p<0.01). In conclusion, genetic and epigenetic changes contribute to variation in SOST expression in human bone. Our data also indicate that these variations may be related to the severity of OI.

Molecular analysis of Berardinelli-Seip congenital lipodystrophy in Oman: evidence for multiple loci.

Congenital generalized lipodystrophy (CGL) is a rare disorder characterized by the absence of body fat and insulin resistance and accompanied by other features, including acanthosis nigricans, organomegaly, hyperandrogenism, and diabetes. We have examined case subjects from 11 families in Oman with CGL. All subjects were the progeny of consanguineous marriages; therefore, a homozygosity mapping strategy was used to investigate the reported loci, 11q13 and 9q34. Three subjects could be linked to 11q13, and mutations were found within the seipin gene. An additional eight subjects were linked to 9q34, but the locus was in a 9-cM interval with no known microsatellites, so further fine mapping was not possible. However, two sibships (four subjects) did not map to either locus, raising the possibility of more than two lipodystrophy loci within the Oman population.

Prevalence of mutations in AGPAT2 among human lipodystrophies.

Berardinelli-Seip congenital lipodystrophy (BSCL) is a heterogeneous genetic disease characterized by near absence of adipose tissue and severe insulin resistance. We have previously identified mutations in the seipin gene in a subset of our patients' cohort. Recently, disease-causing mutations in AGPAT2 have been reported in BSCL patients. In this study, we have performed mutation screening in AGPAT2 and the related AGPAT1 in patients with BSCL or other forms of lipodystrophy who have no detectable mutation in the seipin gene. We found 38 BSCL patients from 30 families with mutations in AGPAT2. Three of the known mutations were frequently found in our families. Of the eight new alterations, six are null mutations and two are missense mutations (Glu172Lys and Ala238Gly). All the patients harboring AGPAT2 mutations presented with typical features of BSCL. We did not find mutations in patients with other forms of lipodystrophies, including the syndromes of Lawrence, Dunnigan, and Barraquer-Simons, or with type A insulin resistance. In conclusion, mutations in the seipin gene and AGPAT2 are confined to the BSCL phenotype. Because we found mutations in 92 of the 94 BSCL patients studied, the seipin gene and AGPAT2 are the two major genes involved in the etiology of BSCL.

Polymorphisms in type II SH2 domain-containing inositol 5-phosphatase (INPPL1, SHIP2) are associated with physiological abnormalities of the metabolic syndrome.

Type II SH2 domain-containing inositol 5-phosphatase (INPPL1, or SHIP2) plays an important role in the control of insulin sensitivity. INPPL1 mutations affecting gene function have been found in rat models of type 2 diabetes and hypertension and in type 2 diabetic patients. We investigated the influence of nucleotide variation in INPPL1 on components of the metabolic syndrome. Following comprehensive resequencing of the gene, we genotyped 12 informative polymorphisms in 1,304 individuals from 424 British type 2 diabetes families that were characterized for several metabolic phenotypes. We have found highly significant associations of single nucleotide polymorphisms (SNPs) and haplotypes of INPPL1 with hypertension as well as with other components of the metabolic syndrome. In a cohort of 905 French type 2 diabetic patients, we found evidence of association of INPPL1 SNPs with the presence of hypertension. We conclude that INPPL1 variants may impact susceptibility to disease and/or to subphenotypes involved in the metabolic syndrome in some diabetic patients.

Wolcott-Rallison Syndrome: clinical, genetic, and functional study of EIF2AK3 mutations and suggestion of genetic heterogeneity.

Wolcott-Rallison syndrome (WRS) is a rare autosomal-recessive disorder characterized by the association of permanent neonatal or early-infancy insulin-dependent diabetes, multiple epiphyseal dysplasia and growth retardation, and other variable multisystemic clinical manifestations. Based on genetic studies of two inbred families, we previously identified the gene responsible for this disorder as EIF2AK3, the pancreatic eukaryotic initiation factor 2alpha (eIF2alpha) kinase. Here, we have studied 12 families with WRS, totalling 18 cases. With the exception of one case, all patients carried EIF2AK3 mutations resulting in truncated or missense versions of the protein. Exclusion of EIF2AK3 mutations in the one patient case was confirmed by both linkage and sequence data. The activities of missense versions of EIF2AK3 were characterized in vivo and in vitro and found to have a complete lack of activity in four mutant proteins and residual kinase activity in one. Remarkably, the onset of diabetes was relatively late (30 months) in the patient expressing the partially defective EIF2AK3 mutant and in the patient with no EIF2AK3 involvement (18 months) compared with other patients (<6 months). The patient with no EIF2AK3 involvement did not have any of the other variable clinical manifestations associated with WRS, which supports the idea that the genetic heterogeneity between this variant form of WRS and EIF2AK3 WRS correlates with some clinical heterogeneity.

Deep sequencing of the Trypanosoma cruzi GP63 surface proteases reveals diversity and diversifying selection among chronic and congenital Chagas disease patients.

BACKGROUND: Chagas disease results from infection with the diploid protozoan parasite Trypanosoma cruzi. T. cruzi is highly genetically diverse, and multiclonal infections in individual hosts are common, but little studied. In this study, we explore T. cruzi infection multiclonality in the context of age, sex and clinical profile among a cohort of chronic patients, as well as paired congenital cases from Cochabamba, Bolivia and Goias, Brazil using amplicon deep sequencing technology. METHODOLOGY/ PRINCIPAL FINDINGS: A 450bp fragment of the trypomastigote TcGP63I surface protease gene was amplified and sequenced across 70 chronic and 22 congenital cases on the Illumina MiSeq platform. In addition, a second, mitochondrial target--ND5--was sequenced across the same cohort of cases. Several million reads were generated, and sequencing read depths were normalized within patient cohorts (Goias chronic, n = 43, Goias congenital n = 2, Bolivia chronic, n = 27; Bolivia congenital, n = 20), Among chronic cases, analyses of variance indicated no clear correlation between intra-host sequence diversity and age, sex or symptoms, while principal coordinate analyses showed no clustering by symptoms between patients. Between congenital pairs, we found evidence for the transmission of multiple sequence types from mother to infant, as well as widespread instances of novel genotypes in infants. Finally, non-synonymous to synonymous (dn:ds) nucleotide substitution ratios among sequences of TcGP63Ia and TcGP63Ib subfamilies within each cohort provided powerful evidence of strong diversifying selection at this locus. CONCLUSIONS/SIGNIFICANCE: Our results shed light on the diversity of parasite DTUs within each patient, as well as the extent to which parasite strains pass between mother and foetus in congenital cases. Although we were unable to find any evidence that parasite diversity accumulates with age in our study cohorts, putative diversifying selection within members of the TcGP63I gene family suggests a link between genetic diversity within this gene family and survival in the mammalian host.

Lipid-related genes and myocardial infarction in 4685 cases and 3460 controls: discrepancies between genotype, blood lipid concentrations, and coronary disease risk.

BACKGROUND: Blood lipid concentrations are causally related to the risk of coronary heart disease (CHD). Various associations between CHD risk and genes that moderately affect plasma lipid levels have been described, but previous studies have typically involved too few 'cases' to assess these associations reliably. METHODS: The present study involves 4685 cases of myocardial infarction (MI) and 3460 unrelated controls without diagnosed cardiovascular disease. Six polymorphisms of four 'lipid-related' genes were genotyped. RESULTS: For the apolipoprotein E epsilon2/epsilon3/epsilon4 polymorphism, the average increase in the plasma ratio of apolipoprotein B to apolipoprotein A(1) (apoB/apoA(1) ratio) among controls was 0.082 (s.e. 0.007) per stepwise change from epsilon3/epsilon2 to epsilon3/epsilon3 to epsilon3/epsilon4 genotype (trend P < 0.0001). The case-control comparison yielded a risk ratio for MI of 1.16 (95% CI: 1.06, 1.27; P = 0.001) per stepwise change in these genotypes. But, this risk ratio was not as extreme as would have been expected from the corresponding differences in plasma apoB/apoA(1) ratio between genotypes. Hence, following adjustment for the measured level of the plasma apoB/apoA(1) ratio, the direction of the risk ratio per stepwise change reversed to 0.83 (95% CI: 0.74, 0.92; P < 0.001). Similarly, for the apolipoprotein B Asn4311Ser and Thr71Ile polymorphisms, genotypes associated with more adverse plasma apolipoprotein concentrations were associated with significantly lower risk of MI after adjustment for the apoB/apoA(1) ratio. The B2 allele of the cholesteryl ester transfer protein TaqIb polymorphism was associated with a significantly lower plasma apoB/apoA(1) ratio, but with no significant difference in the risk of MI. Finally, the lipoprotein lipase Asn291Ser and T4509C (PvuII) polymorphisms did not produce clear effects on either the plasma apoB/apoA(1) ratio or the risk of MI. CONCLUSIONS: It remains unresolved why some of these genetic factors that produce lifelong effects on plasma lipid concentrations have significantly less than the correspondingly expected effects on CHD rates in adult life.