Accurate assignment of disease liability to genetic variants using only population data.

PURPOSE: The growing size of public variant repositories prompted us to test the accuracy of pathogenicity prediction of DNA variants using population data alone. METHODS: Under the a priori assumption that the ratio of the prevalence of variants in healthy population vs that in affected populations form 2 distinct distributions (pathogenic and benign), we used a Bayesian method to assign probability to a variant belonging to either distribution. RESULTS: The approach, termed Bayesian prevalence ratio (BayPR), accurately parsed 300 of 313 expertly curated CFTR variants: 284 of 296 pathogenic/likely pathogenic variants in 1 distribution and 16 of 17 benign/likely benign variants in another. BayPR produced an area under the receiver operating characteristic curve of 0.99 for 103 functionally confirmed missense CFTR variants, which is equal to or exceeds 10 commonly used algorithms (area under the receiver operating characteristic curve range = 0.54-0.99). Application of BayPR to expertly curated variants in 8 genes associated with 7 Mendelian conditions led to the assignment of a disease-causing probability of ≥80% to 1350 of 1374 (98.3%) pathogenic/likely pathogenic variants and of ≤20% to 22 of 23 (95.7%) benign/likely benign variants. CONCLUSION: Irrespective of the variant type or functional effect, the BayPR approach provides probabilities of pathogenicity for DNA variants responsible for Mendelian disorders using only the variant counts in affected and unaffected population samples.

Functional Assays Are Essential for Interpretation of Missense Variants Associated with Variable Expressivity.

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 <10% wild-type CFTR (WT-CFTR) function, a conservative threshold for the development of life-limiting CF lung disease, and five variants had moderately decreased function (10% to ∼25% WT-CFTR). The remaining three variants in this group unexpectedly had >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.

Copy-Number Variation of the Glucose Transporter Gene SLC2A3 and Congenital Heart Defects in the 22q11.2 Deletion Syndrome.

The 22q11.2 deletion syndrome (22q11DS; velocardiofacial/DiGeorge syndrome; VCFS/DGS) is the most common microdeletion syndrome and the phenotypic presentation is highly variable. Approximately 65% of individuals with 22q11DS have a congenital heart defect (CHD), mostly of the conotruncal type, and/or an aortic arch defect. The etiology of this phenotypic variability is not currently known. We hypothesized that copy-number variants (CNVs) outside the 22q11.2 deleted region might increase the risk of being born with a CHD in this sensitized population. Genotyping with Affymetrix SNP Array 6.0 was performed on two groups of subjects with 22q11DS separated by time of ascertainment and processing. CNV analysis was completed on a total of 949 subjects (cohort 1, n = 562; cohort 2, n = 387), 603 with CHDs (cohort 1, n = 363; cohort 2, n = 240) and 346 with normal cardiac anatomy (cohort 1, n = 199; cohort 2, n = 147). Our analysis revealed that a duplication of SLC2A3 was the most frequent CNV identified in the first cohort. It was present in 18 subjects with CHDs and 1 subject without (p = 3.12 × 10(-3), two-tailed Fisher's exact test). In the second cohort, the SLC2A3 duplication was also significantly enriched in subjects with CHDs (p = 3.30 × 10(-2), two-tailed Fisher's exact test). The SLC2A3 duplication was the most frequent CNV detected and the only significant finding in our combined analysis (p = 2.68 × 10(-4), two-tailed Fisher's exact test), indicating that the SLC2A3 duplication might serve as a genetic modifier of CHDs and/or aortic arch anomalies in individuals with 22q11DS.

Rare copy number variants and congenital heart defects in the 22q11.2 deletion syndrome.

The 22q11.2 deletion syndrome (22q11DS; velocardiofacial/DiGeorge syndrome; VCFS/DGS; MIM #192430; 188400) is the most common microdeletion syndrome. The phenotypic presentation of 22q11DS is highly variable; approximately 60-75 % of 22q11DS patients have been reported to have a congenital heart defect (CHD), mostly of the conotruncal type, and/or aortic arch defect. The etiology of the cardiac phenotypic variability is not currently known for the majority of patients. We hypothesized that rare copy number variants (CNVs) outside the 22q11.2 deleted region may modify the risk of being born with a CHD in this sensitized population. Rare CNV analysis was performed using Affymetrix SNP Array 6.0 data from 946 22q11DS subjects with CHDs (n = 607) or with normal cardiac anatomy (n = 339). Although there was no significant difference in the overall burden of rare CNVs, an overabundance of CNVs affecting cardiac-related genes was detected in 22q11DS individuals with CHDs. When the rare CNVs were examined with regard to gene interactions, specific cardiac networks, such as Wnt signaling, appear to be overrepresented in 22q11DS CHD cases but not 22q11DS controls with a normal heart. Collectively, these data suggest that CNVs outside the 22q11.2 region may contain genes that modify risk for CHDs in some 22q11DS patients.

The impact of chromosomal microarray on clinical management: a retrospective analysis.

PURPOSE: Chromosomal microarray has been widely adopted as the first-tier clinical test for individuals with multiple congenital anomalies, developmental delay, intellectual disability, and autism spectrum disorders. Although chromosomal microarray has been extensively shown to provide a higher diagnostic yield than conventional cytogenetic methods, some health insurers refuse to provide coverage for this test, claiming that it is experimental and does not affect patients' clinical management. METHODS: We retrospectively reviewed the electronic medical records of all patients who had abnormal chromosomal microarray findings reported by our laboratory over a 3-year period and quantified the management recommendations made in response to these results. RESULTS: Abnormal chromosomal microarray findings were reported for 12.7% of patients (227/1,780). For patients with clinical follow-up notes available, these results had management implications for 54.5% of patients in the entire abnormal cohort (102/187) and for 42.1% of patients referred for isolated neurodevelopmental disorders (16/38). Recommendations included pharmacological treatment, cancer-related screening or exclusion of screening, contraindications, and referrals for further evaluation. CONCLUSION: These results empirically demonstrate the clinical utility of chromosomal microarray by providing evidence that management was directly affected for the majority of patients in our cohort with abnormal chromosomal microarray findings.

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.

A palindrome-mediated recurrent translocation with 3:1 meiotic nondisjunction: the t(8;22)(q24.13;q11.21).

Palindrome-mediated genomic instability has been associated with chromosomal translocations, including the recurrent t(11;22)(q23;q11). We report a syndrome characterized by extremity anomalies, mild dysmorphia, and intellectual impairment caused by 3:1 meiotic segregation of a previously unrecognized recurrent palindrome-mediated rearrangement, the t(8;22)(q24.13;q11.21). There are at least ten prior reports of this translocation, and nearly identical PATRR8 and PATRR22 breakpoints were validated in several of these published cases. PCR analysis of sperm DNA from healthy males indicates that the t(8;22) arises de novo during gametogenesis in some, but not all, individuals. Furthermore, demonstration that de novo PATRR8-to-PATRR11 translocations occur in sperm suggests that palindrome-mediated translocation is a universal mechanism producing chromosomal rearrangements.

CFTR transcription defects in pancreatic sufficient cystic fibrosis patients with only one mutation in the coding region of CFTR.

BACKGROUND: Patients with cystic fibrosis (CF) manifest a multisystem disease due to deleterious mutations in each gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). However, the role of dysfunctional CFTR is uncertain in individuals with mild forms of CF (ie, pancreatic sufficiency) and mutation in only one CFTR gene. METHODS: Eleven pancreatic sufficient (PS) CF patients with only one CFTR mutation identified after mutation screening (three patients), mutation scanning (four patients) or DNA sequencing (four patients) were studied. Bi-directional sequencing of the coding region of CFTR was performed in patients who had mutation screening or scanning. If a second CFTR mutation was not identified, CFTR mRNA transcripts from nasal epithelial cells were analysed to determine if any PS-CF patients harboured a second CFTR mutation that altered RNA expression. RESULTS: Sequencing of the coding regions of CFTR identified a second deleterious mutation in five of the seven patients who previously had mutation screening or mutation scanning. Five of the remaining six patients with only one deleterious mutation identified in the coding region of one CFTR gene had a pathologic reduction in the amount of RNA transcribed from their other CFTR gene (8.4-16% of wild type). CONCLUSIONS: These results show that sequencing of the coding region of CFTR followed by analysis of CFTR transcription could be a useful diagnostic approach to confirm that patients with mild forms of CF harbour deleterious alterations in both CFTR genes.

The Use of High-Density SNP Array to Map Homozygosity in Consanguineous Families to Efficiently Identify Candidate Genes: Application to Woodhouse-Sakati Syndrome.

Two consanguineous Qatari siblings presented for evaluation: a 17-4/12-year-old male with hypogonadotropic hypogonadism, alopecia, intellectual disability, and microcephaly and his 19-year-old sister with primary amenorrhea, alopecia, and normal cognition. Both required hormone treatment to produce secondary sex characteristics and pubertal development beyond Tanner 1. SNP array analysis of both probands was performed to detect shared regions of homozygosity which may harbor homozygous mutations in a gene causing their common features of abnormal pubertal development, alopecia, and variable cognitive delay. Our patients shared multiple homozygous genomic regions; ten shared regions were >1 Mb in length and constituted 0.99% of the genome. DCAF17, encoding a transmembrane nuclear protein of uncertain function, was the only gene identified in a homozygous region known to cause hypogonadotropic hypogonadism. DCAF17 mutations are associated with Woodhouse-Sakati syndrome, a rare disorder characterized by alopecia, hypogonadotropic hypogonadism, sensorineural hearing loss, diabetes mellitus, and extrapyramidal movements. Sequencing of the coding exons and flanking intronic regions of DCAF17 in the proband revealed homozygosity for a previously described founder mutation (c.436delC). Targeted DCAF17 sequencing of his affected sibling revealed the same homozygous mutation. This family illustrates the utility of SNP array testing in consanguineous families to efficiently and inexpensively identify regions of genomic homozygosity in which genetic candidates for recessive conditions can be identified.

Analysis of the t(3;8) of hereditary renal cell carcinoma: a palindrome-mediated translocation.

It has emerged that palindrome-mediated genomic instability generates DNA-based rearrangements. The presence of palindromic AT-rich repeats (PATRRs) at the translocation breakpoints suggested a palindrome-mediated mechanism in the generation of several recurrent constitutional rearrangements: the t(11;22), t(17;22), and t(8;22). To date, all reported PATRR-mediated translocations include the PATRR on chromosome 22 (PATRR22) as a translocation partner. Here, the constitutional rearrangement, t(3;8)(p14.2;q24.1), segregating with renal cell carcinoma in two families, is examined. The chromosome 8 breakpoint lies in PATRR8 in the first intron of the RNF139 (TRC8) gene, whereas the chromosome 3 breakpoint is located in an AT-rich palindromic sequence in intron 3 of the FHIT gene (PATRR3). Thus, the t(3;8) is the first PATRR-mediated, recurrent, constitutional translocation that does not involve PATRR22. Furthermore, we detect de novo translocations similar to the t(11;22) and t(8;22), involving PATRR3 in normal sperm. The breakpoint on chromosome 3 is in proximity to FRA3B, the most common fragile site in the human genome and a site of frequent deletions in tumor cells. However, the lack of involvement of PATRR3 sequence in numerous FRA3B-related deletions suggests that there are several different DNA sequence-based etiologies responsible for chromosome 3p14.2 genomic rearrangements.

A candidate gene approach to identify modifiers of the palatal phenotype in 22q11.2 deletion syndrome patients.

OBJECTIVE: Palatal anomalies are one of the identifying features of 22q11.2 deletion syndrome (22q11.2DS) affecting about one third of patients. To identify genetic variants that increase the risk of cleft or palatal anomalies in 22q11.2DS patients, we performed a candidate gene association study in 101 patients with 22q11.2DS genotyped with the Affymetrix genome-wide human SNP array 6.0. METHODS: Patients from Children's Hospital of Philadelphia, USA and Wilhelmina Children's Hospital Utrecht, The Netherlands were stratified based on palatal phenotype (overt cleft, submucosal cleft, bifid uvula). SNPs in 21 candidate genes for cleft palate were analyzed for genotype-phenotype association. In addition, TBX1 sequencing was carried out. Quality control and association analyses were conducted using the software package PLINK. RESULTS: Genotype and phenotype data of 101 unrelated patients (63 non-cleft subjects (62.4%), 38 cleft subjects (37.6%)) were analyzed. A Total of 39 SNPs on 10 genes demonstrated a p-value ≤0.05 prior to correction. The most significant SNPs were found on FGF10. However none of the SNPs remained significant after correcting for multiple testing. CONCLUSIONS: Although these results are promising, analysis of additional samples will be required to confirm that variants in these regions influence risk for cleft palate or palatal anomalies in 22q11.2DS patients.

Mutations in the beta-subunit of the epithelial Na+ channel in patients with a cystic fibrosis-like syndrome.

Cystic fibrosis (CF) is an autosomal recessive disorder of Cl(-) and Na(+) transport. The vast majority of CF patients have deleterious mutations in an epithelial Cl(-) channel called the CF transmembrane conductance regulator (CFTR). In contrast, defects in the epithelial Na(+) channel (SCNN1) have been associated with phenotypes dominated by renal disease (systemic pseudohypoaldosteronism type I and Liddle syndrome). We report two non-classic CF patients without CFTR mutations who have novel deleterious mutations in the beta-subunits of SCNN1 in the absence of overt renal disease.