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Ornithine transcarbamylase deficiency (OTCD) is an X-linked inborn error caused by loss of function variants in the OTC gene typically associated with severe neonatal hyperammonemia. Rare examples of late-onset OTCD have also been described. Here, we describe an OTC promoter variant, c.-106CA, in a conserved HNF4a binding site, identified in two male siblings in Family 1 whose first and only recognized episodes of severe hyperammonemia occurred at ages 14 and 39 years, respectively. We identified the same OTC variant segregating in a large family with late-onset OTCD with variable expressivity (Family 2). We show that this OTC promoter variant reduces expression5-fold in a dual-luciferase assay that tests promoter function. Addition of an upstream OTC enhancer increases expression of both the wild type and the c.-106CA variant promoter constructs5-fold with the mutant promoter still about fourfold lower than the wild type. Thus, in both contexts, the promoter variant results in substantially lower OTC expression. Under normal demand on urea cycle function, OTC expression in hemizygous males, although reduced, is sufficient to meet the demand for waste nitrogen excretion. However, in response to severe metabolic stress with attendant increased requirements on urea cycle function, the impaired promoter function results in inadequate OTC expression with resultant hyperammonemia. In the absence of precipitating events, hemizygotes with this allele are asymptomatic, explaining the late age of onset of hyperammonemia in affected individuals and the incomplete penetrance observed in some individuals in Family 2.

Protein translation is a highly regulated process involving the interaction of numerous genes on every component of the protein translation machinery. Upregulated protein translation is a hallmark of cancer and is implicated in autism spectrum disorder, but the risks of developing each disease do not appear to be correlated with one another. In this study we identified two siblings from the NIH Undiagnosed Diseases Program with loss of function variants in PUS7, a gene previously implicated in the regulation of total protein translation. These patients exhibited a neurodevelopmental phenotype including autism spectrum disorder in the proband. Both patients also had features of Lesch-Nyhan syndrome, including hyperuricemia and self-injurious behavior, but without pathogenic variants in HPRT1. Patient fibroblasts demonstrated upregulation of protein synthesis, including elevated MYC protein, but did not exhibit increased rates of cell proliferation. Interestingly, the dysregulation of protein translation also resulted in mildly decreased levels of HPRT1 protein suggesting an association between dysregulated protein translation and the LNS-like phenotypic findings. These findings strengthen the correlation between neurodevelopmental disease, particularly autism spectrum disorders, and the rate of protein translation.

Rationale: The advent of precision treatment for cystic fibrosis using small-molecule therapeutics has created a need to estimate potential clinical improvements attributable to increases in cystic fibrosis transmembrane conductance regulator (CFTR) function. Objectives: To derive CFTR function of a variety of CFTR genotypes and correlate with key clinical features (sweat chloride concentration, pancreatic exocrine status, and lung function) to develop benchmarks for assessing response to CFTR modulators. Methods: CFTR function assigned to 226 unique CFTR genotypes was correlated with the clinical data of 54,671 individuals enrolled in the Clinical and Functional Translation of CFTR (CFTR2) project. Cross-sectional FEV(1)% predicted measurements were plotted by age at which measurement was obtained. Shifts in sweat chloride concentration and lung function reported in CFTR modulator trials were compared with function–phenotype correlations to assess potential efficacy of therapies. Measurements and Main Results: CFTR genotype function exhibited a logarithmic relationship with each clinical feature. Modest increases in CFTR function related to differing genotypes were associated with clinically relevant improvements in cross-sectional FEV(1)% predicted over a range of ages (6–82 yr). Therapeutic responses to modulators corresponded closely to predictions from the CFTR2-derived relationship between CFTR genotype function and phenotype. Conclusions: Increasing CFTR function in individuals with severe disease will have a proportionally greater effect on outcomes than similar increases in CFTR function in individuals with mild disease and should reverse a substantial fraction of the disease process. This study provides reference standards for clinical outcomes that may be achieved by increasing CFTR function.

Missense DNA variants have variable effects upon protein function. Consequently, interpreting their pathogenicity is challenging, especially when they are associated with disease variability. To determine the degree to which functional assays inform interpretation, we analyzed 48 CFTR missense variants associated with variable expressivity of cystic fibrosis (CF). We assessed function in a native isogenic context by evaluating CFTR mutants that were stably expressed in the genome of a human airway cell line devoid of endogenous CFTR expression. 21 of 29 variants associated with full expressivity of the CF phenotype generated 25% WT-CFTR function; two were higher than 75% WT-CFTR. As expected, 14 of 19 variants associated with partial expressivity of CF had >25% WT-CFTR function; however, four had minimal to no effect on CFTR function (>75% WT-CFTR). Thus, 6 of 48 (13%) missense variants believed to be disease causing did not alter CFTR function. Functional studies substantially refined pathogenicity assignment with expert annotation and criteria from the American College of Medical Genetics and Genomics and Association for Molecular Pathology. However, four algorithms (CADD, REVEL, SIFT, and PolyPhen-2) could not differentiate between variants that caused severe, moderate, or minimal reduction in function. In the setting of variable expressivity, these results indicate that functional assays are essential for accurate interpretation of missense variants and that current prediction tools should be used with caution.

Analysis of the functional consequences and treatment response of rare CFTR variants is challenging due to the limited availability of primary airways cells.A Flp recombination target (FRT) site for stable expression of CFTR was incorporated into an immortalized CF bronchial epithelial cell line (CFBE41o-). CFTR cDNA was integrated into the FRT site. Expression was evaluated by western blotting and confocal microscopy and function measured by short circuit current. RNA sequencing was used to compare the transcriptional profile of the resulting CF8Flp cell line to primary cells and tissues.Functional CFTR was expressed from integrated cDNA at the FRT site of the CF8Flp cell line at levels comparable to that seen in native airway cells. CF8Flp cells expressing WT-CFTR have a stable transcriptome comparable to that of primary cultured airway epithelial cells, including genes that play key roles in CFTR pathways.CF8Flp cells provide a viable substitute for primary CF airway cells for the analysis of CFTR variants in a native context.

Elevated sweat chloride levels, failure to thrive (FTT), and lung disease are characteristic features of cystic fibrosis (CF, OMIM #219700). Here we describe variants in CA12 encoding carbonic anhydrase XII in two pedigrees exhibiting CF-like phenotypes. Exome sequencing of a white American adult diagnosed with CF due to elevated sweat chloride, recurrent hyponatremia, infantile FTT and lung disease identified deleterious variants in each CA12 gene: c.908-1 G>A in a splice acceptor and a novel frameshift insertion c.859_860insACCT. In an unrelated consanguineous Omani family, two children with elevated sweat chloride, infantile FTT, and recurrent hyponatremia were homozygous for a novel missense variant (p.His121Gln). Deleterious CFTR variants were absent in both pedigrees. CA XII protein was localized apically in human bronchiolar epithelia and basolaterally in the reabsorptive duct of human sweat glands. Respiratory epithelial cell RNA from the adult proband revealed only aberrant CA12 transcripts and in vitro analysis showed greatly reduced CA XII protein. Studies of ion transport across respiratory epithelial cells in vivo and in culture revealed intact CFTR-mediated chloride transport in the adult proband. CA XII protein bearing either p.His121Gln or a previously identified p.Glu143Lys missense variant localized to the basolateral membranes of polarized Madin-Darby canine kidney (MDCK) cells, but enzyme activity was severely diminished when assayed at physiologic concentrations of extracellular chloride. Our findings indicate that loss of CA XII function should be considered in individuals without CFTR mutations who exhibit CF-like features in the sweat gland and lung.

CFTR modulators have revolutionized the treatment of individuals with cystic fibrosis (CF) by improving the function of existing protein. Unfortunately, almost half of the disease-causing variants in CFTR are predicted to introduce premature termination codons (PTC) thereby causing absence of full-length CFTR protein. We hypothesized that a subset of nonsense and frameshift variants in CFTR allow expression of truncated protein that might respond to FDA-approved CFTR modulators. To address this concept, we selected 26 PTC-generating variants from four regions of CFTR and determined their consequences on CFTR mRNA, protein and function using intron-containing minigenes expressed in 3 cell lines (HEK293, MDCK and CFBE41o-) and patient-derived conditionally reprogrammed primary nasal epithelial cells. The PTC-generating variants fell into five groups based on RNA and protein effects. Group A (reduced mRNA, immature (core glycosylated) protein, function 1% (n = 5)), Group D (reduced mRNA, mature protein, function >1% (n = 5)) and Group E (aberrant RNA splicing, mature protein, function > 1% (n = 1)) variants responded to modulators. Increasing mRNA level by inhibition of NMD led to a significant amplification of modulator effect upon a Group D variant while response of a Group A variant was unaltered. Our work shows that PTC-generating variants should not be generalized as genetic ‘nulls’ as some may allow generation of protein that can be targeted to achieve clinical benefit., Author summary The development of variant specific modulators that correct dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) protein is an excellent example of precision medicine. Currently there is no molecular treatment available for individuals with cystic fibrosis (CF) carrying nonsense or frameshift variants because such variants introduce a premature termination codon (PTC), and are not expected to produce CFTR protein. We have performed a systematic study of nonsense and frameshift variants located in four regions of CFTR that we postulated should have varying effects on mRNA stability, protein production, and/or function. Using primary nasal cells and three different cell line models stably expressing CFTR expression mini-genes (EMGs), we report molecular consequences of 26 PTC-generating variants in CFTR, and identify which variants allow generation of CFTR protein that is responsive to currently available modulator therapies and which require alternative therapeutic approaches.

Treatment of individuals with cystic fibrosis (CF) has been transformed by small molecule therapies that target select pathogenic variants in the CF transmembrane conductance regulator (CFTR). To expand treatment eligibility, we stably expressed 43 rare missense CFTR variants associated with moderate CF from a single site in the genome of human CF bronchial epithelial (CFBE41o-) cells. The magnitude of drug response was highly correlated with residual CFTR function for the potentiator ivacaftor, the corrector lumacaftor, and ivacaftor-lumacaftor combination therapy. Response of a second set of 16 variants expressed stably in Fischer rat thyroid (FRT) cells showed nearly identical correlations. Subsets of variants were identified that demonstrated statistically significantly higher responses to specific treatments. Furthermore, nearly all variants studied in CFBE cells (40 of 43) and FRT cells (13 of 16) demonstrated greater response to ivacaftor-lumacaftor combination therapy than either modulator alone. Together, these variants represent 87% of individuals in the CFTR2 database with at least 1 missense variant. Thus, our results indicate that most individuals with CF carrying missense variants are (a) likely to respond modestly to currently available modulator therapy, while a small fraction will have pronounced responses, and (b) likely to derive the greatest benefit from combination therapy.

With over 1,900 variants reported in the cystic fibrosis transmembrane conductance regulator (CFTR), enhanced understanding of cystic fibrosis (CF) genotype-phenotype correlation represents an important and expanding area of research. The potentiator Ivacaftor has proven an effective treatment for a subset of individuals carrying missense variants, particularly those that impact CFTR gating. Therapeutic efforts have recently focused on correcting the basic defect resulting from the common F508del variant, as well as many less frequent missense alleles. Modest enhancement of F508del-CFTR function has been achieved by combining Ivacaftor with Lumacaftor, a compound that aids maturational processing of misfolded CFTR. Continued development of in silico and in vitro models will facilitate CFTR variant characterization and drug testing, thereby elucidating heterogeneity in the molecular pathogenesis, phenotype, and modulator responsiveness of CF.

Juvenile hyaline fibromatosis (JHF) and infantile systemic hyalinosis (ISH) are rare, autosomal recessive disorders of the connective tissue caused by mutations in the gene encoding the anthrax toxin receptor 2 protein (ANTXR2) located on chromosome 4q21. Characteristically, these conditions present with overlapping clinical features, such as nodules and/or pearly papules, gingival hyperplasia, flexion contractures of the joints, and osteolytic bone defects. The present report describes a pair of sibs and three other JHF/ISH patients whose diagnoses were based on typical clinical manifestations and confirmed by histopathologic analyses and/or molecular analysis. A comparison of ISH and JHF, additional thoughts about new terminology (hyaline fibromatosis syndrome) and a modified grading system are also included.

To the Editor: Amelogenesis imperfecta (AI) are a heterogeneous group of disorders that perturb enamel development. Historically, 14 subtypes have been recognized based on clinical phenotype and mode of inheritance (1). To date, mutations in AMELX, ENAM, KLK4, and MMP20 have been documented in hypoplastic and hypomaturation AI (2–6). However, the genetic basis for most cases of autosomal dominant AI in the Turkish population remained unknown. Recently, mutations underlying a hypocalcified form of AI were reported (7, 8). In the present study, we evaluated eight Turkish kindreds with autosomal dominant hypocalcified AI (ADHCAI). Genetic linkage studies were consistent with linkage to chromosome 8q24.3 in seven families. Sequence analysis identified FAM83H nonsense mutations in all eight families. Eight probands were identified from Dental School Clinics at Ege University, the University of Istanbul and Yeditepe University, Istanbul, Turkey, in accordance with Institutional Review Board approval from the corresponding University and the National Institutes of Health. Available family members received an oral examination and dental radiographs to determine affection status. The presence of AI was established by generalized yellow–brown discoloration of the teeth, hypoplastic appearing enamel and pathological loss of enamel. Affected individuals were diagnosed with ADHCAI based on previously proposed criteria (1) (Fig. 1). Seven kindreds were consistent for autosomal dominant transmission of AI. In an eighth family, only the proband was affected, but the phenotype was consistent with hypocalcified AI and the known AI genes AMELX, ENAM, KLK4, and MMP20 had been excluded based on sequence analysis. A total of 50 individuals were clinically examined, including 26 affected individuals. DNA extraction from peripheral venous blood was performed as previously described (6). DNA extraction from saliva was performed according to the Oragene protocol (DNA Genotek Inc., Ottawa, Ontario, Canada). Fig. 1 Pedigrees, clinical photographs, mutations and haplotype analysis for Turkish families segregating autosomal dominant hypocalcified amelogenesis imperfecta. (a) Family 1 segregating a previously identified mutation, c.1192C>T (p.Q398X). (b) Family ... Primers and conditions used to amplify the FAM83H gene are shown in Table 1. The large approximately 4.8 kb exon 5 was split into eight overlapping amplicons. Polymerase chain reaction (PCR) products were purified using Exo-Sap and sequenced in both directions to minimize sequencing artifacts using ABI Big Dye Terminator chemistry, version 3.1, and an ABI 3730 (Applied Biosystems, Foster City, CA). Table 1 Primers and conditions to amplify FAM83H Six short tandem repeat polymorphism (STRP) markers (D8S1836, D8S15018mg, D8S373, D8S2334, D8S1925 and D8S1926) spanning a 2.35-Mb interval that includes the FAM83H locus were genotyped. Markers were amplified from genomic DNA using fluorescently labeled primers and AmpliTaq Gold PCR Master Mix (Applied Biosystems) according to the manufacturer’s protocol. PCR products were electrophoresed using an ABI 3130 DNA analyzer with size standard GeneScan 400HD Rox. Data were analyzed with GeneMapper ID software, v3.7. Haplotypes were generated based on allele transmission, incorporating sequence variants identified by sequencing. Genotyping of STRP loci surrounding the FAM83H locus in families 1–7 were consistent with genetic linkage to this interval of chromosome eight. As we were unable to amplify the large exon 5 using the published primers (7), primers were redesigned as shown in Table 1 and required a variety of conditions to successfully amplify this region. Three mutations were identified (Fig. 1, Table 2). Affected individuals from family 1 were found to have a C to T substitution at nucleotide position 1192 of the complementary DNA reference sequence (Gen-Bank accession number: {"type":"entrez-nucleotide","attrs":{"text":"NM_198488.3","term_id":"157311634","term_text":"NM_198488.3"}}NM_198488.3), a mutation previously identified in a Korean family (7). A novel c.1366C>T mutation was identified in family 2. This substitution is predicted to be a nonsense mutation, p.Q444X (Fig. 1b). Affected individuals from the remaining six families all had a novel nonsense mutation, p.Q456X, due to a c.1366C>T mutation (Fig. 1c). The finding of a common c.1366C>T mutation in six families (families 3–8) raised the possibility of a mutational hot spot or a founder effect. Based on haplotype analysis, the c.1366C>T mutation appears to have been inherited ‘identical by descent’ from a common ancestor in five of these families with a shared haplotype extending over an interval of 1.8 Mb spanning the FAM83H locus (Fig. 1d). In family 8, the c.1366C>T mutation appears on a different haplotype background. Additionally, the proband was the only affected family member. Mutational analysis confirmed neither parent has a FAM83H mutation. Thus, consistent with the haplotype analysis, this mutation appears to have arisen de novo, but germline mosaicism in one of the parents cannot be excluded. Table 2 Mutations in FAM83H FAM83H mutations were identified in affected individuals from all eight Turkish families studied. With these results, eight FAM83H mutations are now reported (Table 2). Only two mutations, c.1192C>T and c.1366C>T, have been found in more than one kindred. The c.1192C>T mutation reported previously in a Korean family was identified in one Turkish family (family 1) in the current study (7). Based on haplotype analysis, the c.1366C>T mutation identified in six Turkish families reported here appears to have occurred as a founder mutation in five Turkish families (families 3–7) and as an apparent de novo mutation in another Turkish family (family 8, Fig. 1d). Lee et al. (8) also reported a de novo p.E415X FAM83H mutation. Identification of two de novo mutations from a total of eight mutations suggests that the FAM83H gene may have a high mutation rate, which is surprising given that the gene is relatively small (98,126 bp). Consistent with previous findings, all mutations found in these Turkish families were nonsense mutations (7, 8). The finding of only nonsense mutations raises the question of whether the underlying molecular mechanism is haploinsufficiency or a dominant negative effect. Although all reported mutations are in the terminal exon and the corresponding messenger RNAs might be expected to escape nonsense-mediated decay (NMD), there are examples in humans of terminal exon nonsense mutations triggering NMD, notably in COL10A1 (9). If mutant transcripts undergo NMD then haploinsufficiency of FAM83H leads to ADHCAI. If the transcripts escape NMD then the truncated products may interfere with the normal product and produce a phenotype through a dominant negative effect. The finding that all mutations are nonsense mutations that cluster within a 380 amino acid region is more supportive of a dominant negative effect. As mutations are identified in more families, the underlying molecular mechanism may become clearer. FAM83H represents the first gene widely expressed outside of the developing tooth to be associated with AI. Kim et al. (7) demonstrated expression in mouse eye, liver and kidney. We verified expression in human kidney, liver and gingiva (data not shown). The phenotype observed in association with FAM83H mutations indicates that FAM83H functions in mineralization, but its expression in non-mineralized tissues suggests that either it does not directly function in mineralization or it has another function in non-mineralized tissues. Enamel matrix proteins are almost completely removed as enamel crystallites grow and mineralization progresses (10). In both hypomaturation and hypocalcified forms of AI, significant amounts of protein are detected in fully developed enamel (11). It is not surprising, therefore, that mutation in the known enamel proteases, kallikrein 4 and matrix metalloproteinase 20, result in hypomaturation phenotypes (4–6). MMP20 and KLK4 mutations result in autosomal recessive phenotypes, suggesting that haploinsufficiency of these proteinases is sufficient for normal enamel development. Analysis of the predicted protein sequence of FAM83H does not identify obvious homology outside of the FAM83 family. Given that FAM83H mutations cluster within a 380 amino acid region, this region appears to be critical for normal enamel development and calcification. FAM83H mutations were identified in all eight of the Turkish families segregating ADHCAI, suggesting that FAM83H mutations may be responsible for the majority of ADHCAI in Turkey. The generality of the founder effect in the Turkish population remains to be seen.

FAM83H gene mutations are associated with autosomal-dominant hypocalcified amelogenesis imperfecta (ADHCAI), which is typically characterized by enamel having normal thickness and a markedly decreased mineral content. This study tested the hypothesis that there are phenotype and genotype associations in families with FAM83H-associated ADHCAI. Seven families segregating ADHCAI (147 individuals) were evaluated. Phenotyping included clinical, radiographic, histological, and biochemical studies, and genotyping was by mutational analysis. Multiple novel FAM83H mutations were identified, including two 2-bp-deletion mutations, the first non-nonsense mutations identified. Craniofacial deviation from normal was more prevalent in the affected individuals. Affected individuals having truncating FAMH3H mutations of 677 or fewer amino acids presented a generalized ADHCAI phenotype, while those having mutations capable of producing a protein of at least 694 amino acids had a unique and previously unreported phenotype affecting primarily the cervical enamel. This investigation shows that unique phenotypes are associated with specific FAM83H mutations.