The clinical utility of sequencing the entirety of CFTR.

BACKGROUND: Cystic fibrosis (CF) is caused by deleterious variants in each CFTR gene. We investigated the utility of whole-gene CFTR sequencing when fewer than two pathogenic or likely pathogenic (P/LP) variants were detected by conventional testing (sequencing of exons and flanking introns) of CFTR. METHODS: Individuals with features of CF and a CF-diagnostic sweat chloride concentration with zero or one P/LP variants identified by conventional testing enrolled in the CF Mutation Analysis Program (MAP) underwent whole-gene CFTR sequencing. Replication was performed on individuals enrolled in the CF Genome Project (CFGP), followed by phenotype review and interrogation of other genes. RESULTS: Whole-gene sequencing identified a second P/LP variant in 20/43 MAP enrollees (47 %) and 10/22 CFGP enrollees (45 %) who had one P/LP variant after conventional testing. No P/LP variants were detected when conventional testing was negative (MAP: n = 43; CFGP: n = 13). Genome-wide analysis was unable to find an alternative etiology in CFGP participants with fewer than two P/LP CFTR variants and CF could not be confirmed in 91 % following phenotype re-review. CONCLUSIONS: Whole-gene CFTR analysis is beneficial in individuals with one previously-identified P/LP variant and a CF-diagnostic sweat chloride. Negative conventional CFTR testing indicates that the phenotype should be re-evaluated.

Variants in ACTC1 underlie distal arthrogryposis accompanied by congenital heart defects.

Contraction of the human sarcomere is the result of interactions between myosin cross-bridges and actin filaments. Pathogenic variants in genes such as MYH7, TPM1, and TNNI3 that encode parts of the cardiac sarcomere cause muscle diseases that affect the heart, such as dilated cardiomyopathy and hypertrophic cardiomyopathy. In contrast, pathogenic variants in homologous genes such as MYH2, TPM2, and TNNI2 that encode parts of the skeletal muscle sarcomere cause muscle diseases affecting skeletal muscle, such as distal arthrogryposis (DA) syndromes and skeletal myopathies. To date, there have been few reports of genes (e.g., MYH7) encoding sarcomeric proteins in which the same pathogenic variant affects skeletal and cardiac muscle. Moreover, none of the known genes underlying DA have been found to contain pathogenic variants that also cause cardiac abnormalities. We report five families with DA because of heterozygous missense variants in the gene actin, alpha, cardiac muscle 1 (ACTC1). ACTC1 encodes a highly conserved actin that binds to myosin in cardiac and skeletal muscle. Pathogenic variants in ACTC1 have been found previously to underlie atrial septal defect, dilated cardiomyopathy, hypertrophic cardiomyopathy, and left ventricular noncompaction. Our discovery delineates a new DA condition because of variants in ACTC1 and suggests that some functions of ACTC1 are shared in cardiac and skeletal muscle.

Variants in ACTC1 underlie distal arthrogryposis accompanied by congenital heart defects.

Contraction of the human sarcomere is the result of interactions between myosin cross-bridges and actin filaments. Pathogenic variants in genes such as MYH7 , TPM1 , and TNNI3 that encode parts of the cardiac sarcomere cause muscle diseases that affect the heart, such as dilated cardiomyopathy and hypertrophic cardiomyopathy. In contrast, pathogenic variants in homologous genes MYH2 , TPM2 , and TNNI2 , that encode parts of the skeletal muscle sarcomere, cause muscle diseases affecting skeletal muscle, such as the distal arthrogryposis (DA) syndromes and skeletal myopathies. To date, there have been few reports of genes (e.g., MYH7 ) encoding sarcomeric proteins in which the same pathogenic variant affects both skeletal and cardiac muscle. Moreover, none of the known genes underlying DA have been found to contain mutations that also cause cardiac abnormalities. We report five families with DA due to heterozygous missense variants in the gene actin, alpha, cardiac muscle 1 ( ACTC1 ). ACTC1 encodes a highly conserved actin that binds to myosin in both cardiac and skeletal muscle. Mutations in ACTC1 have previously been found to underlie atrial septal defect, dilated cardiomyopathy, hypertrophic cardiomyopathy, and left ventricular noncompaction. Our discovery delineates a new DA condition due to mutations in ACTC1 and suggests that some functions of actin, alpha, cardiac muscle 1 are shared in cardiac and skeletal muscle.

Genetic Modifiers of Cystic Fibrosis Lung Disease Severity: Whole-Genome Analysis of 7,840 Patients.

Rationale: Lung disease is the major cause of morbidity and mortality in persons with cystic fibrosis (pwCF). Variability in CF lung disease has substantial non-CFTR (CF transmembrane conductance regulator) genetic influence. Identification of genetic modifiers has prognostic and therapeutic importance. Objectives: Identify genetic modifier loci and genes/pathways associated with pulmonary disease severity. Methods: Whole-genome sequencing data on 4,248 unique pwCF with pancreatic insufficiency and lung function measures were combined with imputed genotypes from an additional 3,592 patients with pancreatic insufficiency from the United States, Canada, and France. This report describes association of approximately 15.9 million SNPs using the quantitative Kulich normal residual mortality-adjusted (KNoRMA) lung disease phenotype in 7,840 pwCF using premodulator lung function data. Measurements and Main Results: Testing included common and rare SNPs, transcriptome-wide association, gene-level, and pathway analyses. Pathway analyses identified novel associations with genes that have key roles in organ development, and we hypothesize that these genes may relate to dysanapsis and/or variability in lung repair. Results confirmed and extended previous genome-wide association study findings. These whole-genome sequencing data provide finely mapped genetic information to support mechanistic studies. No novel primary associations with common single variants or rare variants were found. Multilocus effects at chr5p13 (SLC9A3/CEP72) and chr11p13 (EHF/APIP) were identified. Variant effect size estimates at associated loci were consistently ordered across the cohorts, indicating possible age or birth cohort effects. Conclusions: This premodulator genomic, transcriptomic, and pathway association study of 7,840 pwCF will facilitate mechanistic and postmodulator genetic studies and the development of novel therapeutics for CF lung disease.

Club cell secretory protein and lung function in children with cystic fibrosis.

BACKGROUND: Club cell secretory protein (CC16) exerts anti-inflammatory functions in lung disease. We sought to determine the relation of serum CC16 deficits and genetic variants that control serum CC16 to lung function among children with cystic fibrosis (CF). METHODS: We used longitudinal data from CF children (EPIC Study) with no positive cultures for Pseudomonas aeruginosa prior to enrollment. Circulating levels of CC16 and an inflammatory score (generated from CRP, SAA, calprotectin, G-CSF) were compared between participants with the lowest and highest FEV1 levels in adolescence (LLF and HLF groups, respectively; N = 130-per-group). Single nucleotide variants (SNVs) in the SCGB1A1, EHF-APIP loci were tested for association with circulating CC16 and with decline of FEV1 and FEV1/FVC% predicted levels between ages 7-16 using mixed models. RESULTS: Compared with the HLF group, the LLF group had lower levels of CC16 (geometric means: 8.2 vs 6.5 ng/ml, respectively; p = 0.0002) and higher levels of the normalized inflammatory score (-0.21 vs 0.21, p = 0.0007). Participants in the lowest CC16 and highest inflammation tertile had the highest odds for having LLF (p<0.0001 for comparison with participants in the highest CC16 and lowest inflammation tertile). Among seven SNVs associated with circulating CC16, the top SNV rs3741240 was associated with decline of FEV1/FVC and, marginally, FEV1 (p = 0.003 and 0.025, respectively; N = 611 participants, 20,801 lung function observations). CONCLUSIONS: Serum CC16 deficits are strongly associated with severity of CF lung disease and their effects are additive with systemic inflammation. The rs3741240 A allele is associated with low circulating CC16 and, possibly, accelerated lung function decline in CF.

Inflammasome Genetic Variants, Macrophage Function, and Clinical Outcomes in Cystic Fibrosis.

Cystic fibrosis (CF) is characterized by chronic airway infection, inflammation, and tissue damage that lead to progressive respiratory failure. NLRP3 and NLRC4 are cytoplasmic pattern recognition receptors that activate the inflammasome, initiating a caspase-1-mediated response. We hypothesized that gain-of-function inflammasome responses are associated with worse outcomes in children with CF. We genotyped nonsynonymous variants in NLRP3 and the NLRC4 pathway from individuals in the EPIC (Early Pseudomonas Infection Control) Observational Study cohort and tested for association with CF outcomes. We generated knockouts of NLRP3 and NLRC4 in human macrophage-like cells and rescued knockouts with wild-type or variant forms of NLRP3 and NLRC4. We identified a SNP in NLRP3, p.(Q705K), that was associated with a higher rate of P. aeruginosa colonization (N = 609; P = 0.01; hazard ratio, 2.3 [Cox model]) and worsened lung function over time as measured by forced expiratory volume in 1 second (N = 445; P = 0.001 [generalized estimating equation]). We identified a SNP in NLRC4, p.(A929S), that was associated with a lower rate of P. aeruginosa colonization as part of a composite of rare variants (N = 405; P = 0.045; hazard ratio, 0.68 [Cox model]) and that was individually associated with protection from lung function decline (P < 0.001 [generalized estimating equation]). Rescue of the NLRP3 knockout with the p.(Q705K) variant produced significantly more IL-1ß in response to NLRP3 stimulation than rescue with the wild type (P = 0.020 [Student's t test]). We identified a subset of children with CF at higher risk of early lung disease progression. Knowledge of these genetic modifiers could guide therapies targeting inflammasome pathways.

Mutations in MYLPF Cause a Novel Segmental Amyoplasia that Manifests as Distal Arthrogryposis.

We identified ten persons in six consanguineous families with distal arthrogryposis (DA) who had congenital contractures, scoliosis, and short stature. Exome sequencing revealed that each affected person was homozygous for one of two different rare variants (c.470G>T [p.Cys157Phe] or c.469T>C [p.Cys157Arg]) affecting the same residue of myosin light chain, phosphorylatable, fast skeletal muscle (MYLPF). In a seventh family, a c.487G>A (p.Gly163Ser) variant in MYLPF arose de novo in a father, who transmitted it to his son. In an eighth family comprised of seven individuals with dominantly inherited DA, a c.98C>T (p.Ala33Val) variant segregated in all four persons tested. Variants in MYLPF underlie both dominant and recessively inherited DA. Mylpf protein models suggest that the residues associated with dominant DA interact with myosin whereas the residues altered in families with recessive DA only indirectly impair this interaction. Pathological and histological exam of a foot amputated from an affected child revealed complete absence of skeletal muscle (i.e., segmental amyoplasia). To investigate the mechanism for this finding, we generated an animal model for partial MYLPF impairment by knocking out zebrafish mylpfa. The mylpfa mutant had reduced trunk contractile force and complete pectoral fin paralysis, demonstrating that mylpf impairment most severely affects limb movement. mylpfa mutant muscle weakness was most pronounced in an appendicular muscle and was explained by reduced myosin activity and fiber degeneration. Collectively, our findings demonstrate that partial loss of MYLPF function can lead to congenital contractures, likely as a result of degeneration of skeletal muscle in the distal limb.

Mutations in the translocon-associated protein complex subunit SSR3 cause a novel congenital disorder of glycosylation.

The translocon-associated protein (TRAP) complex facilitates the translocation of proteins across the endoplasmic reticulum membrane and associates with the oligosaccharyl transferase (OST) complex to maintain proper glycosylation of nascent polypeptides. Pathogenic variants in either complex cause a group of rare genetic disorders termed, congenital disorders of glycosylation (CDG). We report an individual who presented with severe intellectual and developmental disabilities and sensorineural deafness with an unsolved type I CDG, and sought to identify the underlying genetic basis. Exome sequencing identified a novel homozygous variant c.278_281delAGGA [p.Glu93Valfs*7] in the signal sequence receptor 3 (SSR3) subunit of the TRAP complex. Biochemical studies in patient fibroblasts showed the variant destabilized the TRAP complex with a complete loss of SSR3 protein and partial loss of SSR1 and SSR4. Importantly, all subunit levels were corrected by expression of wild-type SSR3. Abnormal glycosylation status in fibroblasts was confirmed using two markers proteins, GP130 and ICAM1. Our findings confirm mutations in SSR3 cause a novel CDG. A novel frameshift variant in the translocon associated protein, SSR3, disrupts the stability of the TRAP complex and causes a novel Congenital Disorder of Glycosylation.

Correction: Whole genome sequencing of extreme phenotypes identifies variants in CD101 and UBE2V1 associated with increased risk of sexually acquired HIV-1.

[This corrects the article DOI: 10.1371/journal.ppat.1006703.].

DPAGT1 Deficiency with Encephalopathy (DPAGT1-CDG): Clinical and Genetic Description of 11 New Patients.

Pathogenic mutations in DPAGT1 cause a rare type of a congenital disorder of glycosylation termed DPAGT1-CDG or, alternatively, a milder version with only myasthenia known as DPAGT1-CMS. Fourteen disease-causing mutations in 28 patients from 10 families have previously been reported to cause the systemic form, DPAGT1-CDG. We here report on another 11 patients from 8 families and add 10 new mutations. Most patients have a very severe disease course, where common findings are pronounced muscular hypotonia, intractable epilepsy, global developmental delay/intellectual disability, and early death. We also present data on three affected females that are young adults and have a somewhat milder, stable disease. Our findings expand both the molecular and clinical knowledge of previously published data but also widen the phenotypic spectrum of DPAGT1-CDG.

Whole genome sequencing of extreme phenotypes identifies variants in CD101 and UBE2V1 associated with increased risk of sexually acquired HIV-1.

Host genetic variation modifying HIV-1 acquisition risk can inform development of HIV-1 prevention strategies. However, associations between rare or intermediate-frequency variants and HIV-1 acquisition are not well studied. We tested for the association between variation in genic regions and extreme HIV-1 acquisition phenotypes in 100 sub-Saharan Africans with whole genome sequencing data. Missense variants in immunoglobulin-like regions of CD101 and, among women, one missense/5' UTR variant in UBE2V1, were associated with increased HIV-1 acquisition risk (p = 1.9x10-4 and p = 3.7x10-3, respectively, for replication). Both of these genes are known to impact host inflammatory pathways. Effect sizes increased with exposure to HIV-1 after adjusting for the independent effect of increasing exposure on acquisition risk. TRIAL REGISTRATION: ClinicalTrials.gov NCT00194519; NCT00557245.

Encephalopathy caused by novel mutations in the CMP-sialic acid transporter, SLC35A1.

Transport of activated nucleotide-sugars into the Golgi is critical for proper glycosylation and mutations in these transporters cause a group of rare genetic disorders termed congenital disorders of glycosylation. We performed exome sequencing on an individual with a profound neurological presentation and identified rare compound heterozygous mutations, p.Thr156Arg and p.Glu196Lys, in the CMP-sialic acid transporter, SLC35A1. Patient primary fibroblasts and serum showed a considerable decrease in the amount of N- and O-glycans terminating in sialic acid. Direct measurement of CMP-sialic acid transport into the Golgi showed a substantial decrease in overall rate of transport. Here we report the identification of the third patient with CMP-sialic acid transporter deficiency, who presented with severe neurological phenotype, but without hematological abnormalities.

Activation of a cryptic splice site in the mitochondrial elongation factor GFM1 causes combined OXPHOS deficiency.

We report the clinical, biochemical, and molecular findings in two brothers with encephalopathy and multi-systemic disease. Abnormal transferrin glycoforms were suggestive of a type I congenital disorder of glycosylation (CDG). While exome sequencing was negative for CDG related candidate genes, the testing revealed compound heterozygous mutations in the mitochondrial elongation factor G gene (GFM1). One of the mutations had been reported previously while the second, novel variant was found deep in intron 6, activating a cryptic splice site. Functional studies demonstrated decreased GFM1 protein levels, suggested disrupted assembly of mitochondrial complexes III and V and decreased activities of mitochondrial complexes I and IV, all indicating combined OXPHOS deficiency.

SRD5A3-CDG: Expanding the phenotype of a congenital disorder of glycosylation with emphasis on adult onset features.

Increasing numbers of congenital disorders of glycosylation (CDG) have been reported recently resulting in an expansion of the phenotypes associated with this group of disorders. SRD5A3 codes for polyprenol reductase which converts polyprenol to dolichol. This is a major pathway for dolichol biosynthesis for N-glycosylation, O-mannosylation, C-mannosylation, and GPI anchor synthesis. We present the features of five individuals (three children and two adults) with mutations in SRD5A3 focusing on the variable eye and skin involvement. We compare that to 13 affected individuals from the literature including five adults allowing us to delineate the features that may develop over time with this disorder including kyphosis, retinitis pigmentosa, and cataracts. © 2016 Wiley Periodicals, Inc.

ALG1-CDG: Clinical and Molecular Characterization of 39 Unreported Patients.

Congenital disorders of glycosylation (CDG) arise from pathogenic mutations in over 100 genes leading to impaired protein or lipid glycosylation. ALG1 encodes a ß1,4 mannosyltransferase that catalyzes the addition of the first of nine mannose moieties to form a dolichol-lipid linked oligosaccharide intermediate required for proper N-linked glycosylation. ALG1 mutations cause a rare autosomal recessive disorder termed ALG1-CDG. To date 13 mutations in 18 patients from 14 families have been described with varying degrees of clinical severity. We identified and characterized 39 previously unreported cases of ALG1-CDG from 32 families and add 26 new mutations. Pathogenicity of each mutation was confirmed based on its inability to rescue impaired growth or hypoglycosylation of a standard biomarker in an alg1-deficient yeast strain. Using this approach we could not establish a rank order comparison of biomarker glycosylation and patient phenotype, but we identified mutations with a lethal outcome in the first two years of life. The recently identified protein-linked xeno-tetrasaccharide biomarker, NeuAc-Gal-GlcNAc2 , was seen in all 27 patients tested. Our study triples the number of known patients and expands the molecular and clinical correlates of this disorder.

Expanding the Molecular and Clinical Phenotype of SSR4-CDG.

Congenital disorders of glycosylation (CDG) are a group of mostly autosomal recessive disorders primarily characterized by neurological abnormalities. Recently, we described a single CDG patient with a de novo mutation in the X-linked gene, Signal Sequence Receptor 4 (SSR4). We performed whole-exome sequencing to identify causal variants in several affected individuals who had either an undifferentiated neurological disorder or unsolved CDG of unknown etiology based on abnormal transferrin glycosylation. We now report eight affected males with either de novo (4) or inherited (4) loss of function mutations in SSR4. Western blot analysis revealed that the mutations caused a complete loss of SSR4 protein. In nearly all cases, the abnormal glycosylation of serum transferrin was only slightly above the accepted normal cutoff range.

The Genetic Basis of Mendelian Phenotypes: Discoveries, Challenges, and Opportunities.

Discovering the genetic basis of a Mendelian phenotype establishes a causal link between genotype and phenotype, making possible carrier and population screening and direct diagnosis. Such discoveries also contribute to our knowledge of gene function, gene regulation, development, and biological mechanisms that can be used for developing new therapeutics. As of February 2015, 2,937 genes underlying 4,163 Mendelian phenotypes have been discovered, but the genes underlying ∼50% (i.e., 3,152) of all known Mendelian phenotypes are still unknown, and many more Mendelian conditions have yet to be recognized. This is a formidable gap in biomedical knowledge. Accordingly, in December 2011, the NIH established the Centers for Mendelian Genomics (CMGs) to provide the collaborative framework and infrastructure necessary for undertaking large-scale whole-exome sequencing and discovery of the genetic variants responsible for Mendelian phenotypes. In partnership with 529 investigators from 261 institutions in 36 countries, the CMGs assessed 18,863 samples from 8,838 families representing 579 known and 470 novel Mendelian phenotypes as of January 2015. This collaborative effort has identified 956 genes, including 375 not previously associated with human health, that underlie a Mendelian phenotype. These results provide insight into study design and analytical strategies, identify novel mechanisms of disease, and reveal the extensive clinical variability of Mendelian phenotypes. Discovering the gene underlying every Mendelian phenotype will require tackling challenges such as worldwide ascertainment and phenotypic characterization of families affected by Mendelian conditions, improvement in sequencing and analytical techniques, and pervasive sharing of phenotypic and genomic data among researchers, clinicians, and families.

Autosomal-Dominant Multiple Pterygium Syndrome Is Caused by Mutations in MYH3.

Multiple pterygium syndrome (MPS) is a phenotypically and genetically heterogeneous group of rare Mendelian conditions characterized by multiple pterygia, scoliosis, and congenital contractures of the limbs. MPS typically segregates as an autosomal-recessive disorder, but rare instances of autosomal-dominant transmission have been reported. Whereas several mutations causing recessive MPS have been identified, the genetic basis of dominant MPS remains unknown. We identified four families affected by dominantly transmitted MPS characterized by pterygia, camptodactyly of the hands, vertebral fusions, and scoliosis. Exome sequencing identified predicted protein-altering mutations in embryonic myosin heavy chain (MYH3) in three families. MYH3 mutations underlie distal arthrogryposis types 1, 2A, and 2B, but all mutations reported to date occur in the head and neck domains. In contrast, two of the mutations found to cause MPS in this study occurred in the tail domain. The phenotypic overlap among persons with MPS, coupled with physical findings distinct from other conditions caused by mutations in MYH3, suggests that the developmental mechanism underlying MPS differs from that of other conditions and/or that certain functions of embryonic myosin might be perturbed by disruption of specific residues and/or domains. Moreover, the vertebral fusions in persons with MPS, coupled with evidence of MYH3 expression in bone, suggest that embryonic myosin plays a role in skeletal development.

Speech and language in a genotyped cohort of individuals with Kabuki syndrome.

Speech and language deficits are commonly associated with Kabuki syndrome. Yet little is known regarding the specific symptomatology of these disorders, preventing use of targeted treatment programs. Here we detail speech and language in 16 individuals with Kabuki syndrome (thirteen with KMT2D mutations, one with a KDM6A mutation, and two mutation-negative cases), aged 4-21 years. The most striking speech deficit was dysarthria, characterised by imprecise consonants, harsh vocal quality, hypernasality, reduced rate and stress, and distorted pitch. Oromotor functioning was also impaired. Delayed, rather than disordered, articulation and phonology was common. Both receptive and expressive language abilities were reduced in the majority and deficits were noted across all language sub-domains (i.e., semantics, syntax, morphology, and pragmatics) with no clear differentiation or specific language profile. Individuals with Kabuki syndrome present with a heterogenous pattern of oromotor, speech, and language deficits. This variability fits with the multisystem nature of the disorder, which may encompass neurological, orofacial structural, hearing, and cognitive deficits, any or all of which may contribute to speech or language impairment. Our results suggest that all individuals with Kabuki syndrome have some level of communication deficit, warranting speech pathology involvement in all cases.