Critical assessment of variant prioritization methods for rare disease diagnosis within the rare genomes project.

BACKGROUND: A major obstacle faced by families with rare diseases is obtaining a genetic diagnosis. The average "diagnostic odyssey" lasts over five years and causal variants are identified in under 50%, even when capturing variants genome-wide. To aid in the interpretation and prioritization of the vast number of variants detected, computational methods are proliferating. Knowing which tools are most effective remains unclear. To evaluate the performance of computational methods, and to encourage innovation in method development, we designed a Critical Assessment of Genome Interpretation (CAGI) community challenge to place variant prioritization models head-to-head in a real-life clinical diagnostic setting. METHODS: We utilized genome sequencing (GS) data from families sequenced in the Rare Genomes Project (RGP), a direct-to-participant research study on the utility of GS for rare disease diagnosis and gene discovery. Challenge predictors were provided with a dataset of variant calls and phenotype terms from 175 RGP individuals (65 families), including 35 solved training set families with causal variants specified, and 30 unlabeled test set families (14 solved, 16 unsolved). We tasked teams to identify causal variants in as many families as possible. Predictors submitted variant predictions with estimated probability of causal relationship (EPCR) values. Model performance was determined by two metrics, a weighted score based on the rank position of causal variants, and the maximum F-measure, based on precision and recall of causal variants across all EPCR values. RESULTS: Sixteen teams submitted predictions from 52 models, some with manual review incorporated. Top performers recalled causal variants in up to 13 of 14 solved families within the top 5 ranked variants. Newly discovered diagnostic variants were returned to two previously unsolved families following confirmatory RNA sequencing, and two novel disease gene candidates were entered into Matchmaker Exchange. In one example, RNA sequencing demonstrated aberrant splicing due to a deep intronic indel in ASNS, identified in trans with a frameshift variant in an unsolved proband with phenotypes consistent with asparagine synthetase deficiency. CONCLUSIONS: Model methodology and performance was highly variable. Models weighing call quality, allele frequency, predicted deleteriousness, segregation, and phenotype were effective in identifying causal variants, and models open to phenotype expansion and non-coding variants were able to capture more difficult diagnoses and discover new diagnoses. Overall, computational models can significantly aid variant prioritization. For use in diagnostics, detailed review and conservative assessment of prioritized variants against established criteria is needed.

Scalable approaches for generating, validating and incorporating data from high-throughput functional assays to improve clinical variant classification.

As the adoption and scope of genetic testing continue to expand, interpreting the clinical significance of DNA sequence variants at scale remains a formidable challenge, with a high proportion classified as variants of uncertain significance (VUSs). Genetic testing laboratories have historically relied, in part, on functional data from academic literature to support variant classification. High-throughput functional assays or multiplex assays of variant effect (MAVEs), designed to assess the effects of DNA variants on protein stability and function, represent an important and increasingly available source of evidence for variant classification, but their potential is just beginning to be realized in clinical lab settings. Here, we describe a framework for generating, validating and incorporating data from MAVEs into a semi-quantitative variant classification method applied to clinical genetic testing. Using single-cell gene expression measurements, cellular evidence models were built to assess the effects of DNA variation in 44 genes of clinical interest. This framework was also applied to models for an additional 22 genes with previously published MAVE datasets. In total, modeling data was incorporated from 24 genes into our variant classification method. These data contributed evidence for classifying 4043 observed variants in over 57,000 individuals. Genetic testing laboratories are uniquely positioned to generate, analyze, validate, and incorporate evidence from high-throughput functional data and ultimately enable the use of these data to provide definitive clinical variant classifications for more patients.

Spectrum of splicing variants in disease genes and the ability of RNA analysis to reduce uncertainty in clinical interpretation.

The complexities of gene expression pose challenges for the clinical interpretation of splicing variants. To better understand splicing variants and their contribution to hereditary disease, we evaluated their prevalence, clinical classifications, and associations with diseases, inheritance, and functional characteristics in a 689,321-person clinical cohort and two large public datasets. In the clinical cohort, splicing variants represented 13% of all variants classified as pathogenic (P), likely pathogenic (LP), or variants of uncertain significance (VUSs). Most splicing variants were outside essential splice sites and were classified as VUSs. Among all individuals tested, 5.4% had a splicing VUS. If RNA analysis were to contribute supporting evidence to variant interpretation, we estimated that splicing VUSs would be reclassified in 1.7% of individuals in our cohort. This would result in a clinically significant result (i.e., P/LP) in 0.1% of individuals overall because most reclassifications would change VUSs to likely benign. In ClinVar, splicing VUSs were 4.8% of reported variants and could benefit from RNA analysis. In the Genome Aggregation Database (gnomAD), splicing variants comprised 9.4% of variants in protein-coding genes; most were rare, precluding unambiguous classification as benign. Splicing variants were depleted in genes associated with dominant inheritance and haploinsufficiency, although some genes had rare variants at essential splice sites or had common splicing variants that were most likely compatible with normal gene function. Overall, we describe the contribution of splicing variants to hereditary disease, the potential utility of RNA analysis for reclassifying splicing VUSs, and how natural variation may confound clinical interpretation of splicing variants.

Applications of artificial intelligence in clinical laboratory genomics.

The transition from analog to digital technologies in clinical laboratory genomics is ushering in an era of "big data" in ways that will exceed human capacity to rapidly and reproducibly analyze those data using conventional approaches. Accurately evaluating complex molecular data to facilitate timely diagnosis and management of genomic disorders will require supportive artificial intelligence methods. These are already being introduced into clinical laboratory genomics to identify variants in DNA sequencing data, predict the effects of DNA variants on protein structure and function to inform clinical interpretation of pathogenicity, link phenotype ontologies to genetic variants identified through exome or genome sequencing to help clinicians reach diagnostic answers faster, correlate genomic data with tumor staging and treatment approaches, utilize natural language processing to identify critical published medical literature during analysis of genomic data, and use interactive chatbots to identify individuals who qualify for genetic testing or to provide pre-test and post-test education. With careful and ethical development and validation of artificial intelligence for clinical laboratory genomics, these advances are expected to significantly enhance the abilities of geneticists to translate complex data into clearly synthesized information for clinicians to use in managing the care of their patients at scale.

Deletions in DNAL1 Cause Primary Ciliary Dyskinesia Across North American Indigenous Populations.

We report 4 cases of primary ciliary dyskinesia in unrelated indigenous North American children caused by identical, homozygous, likely pathogenic deletions in the DNAL1 gene. These shared DNAL1 deletions among dispersed indigenous populations suggest that primary ciliary dyskinesia accounts for more lung disease with bronchiectasis than previously recognized in indigenous North Americans.

Fumarate hydratase variant prevalence and manifestations among individuals receiving germline testing.

BACKGROUND: Germline variants in fumarate hydratase (FH) are associated with autosomal dominant (AD) hereditary leiomyomatosis and renal cell cancer (HLRCC) and autosomal recessive (AR) fumarase deficiency (FMRD). The prevalence and cancer penetrance across different FH variants remain unclear. METHODS: A database containing 120,061 records from individuals undergoing cancer germline testing was obtained. FH variants were classified into 3 categories: AD HLRCC variants, AR FMRD variants, and variants of unknown significance (VUSs). Individuals with variants from these categories were compared with those with negative genetic testing. RESULTS: FH variants were detected in 1.3% of individuals (AD HLRCC, 0.3%; AR FMRD, 0.4%; VUS, 0.6%). The rate of AD HLRCC variants discovered among reportedly asymptomatic individuals without a clear indication for HLRCC testing was 1 in 2668 (0.04%). In comparison with those with negative genetic testing, the renal cell carcinoma (RCC) prevalence was elevated with AD HLRCC variants (17.0% vs 4.5%; P < .01) and VUSs (6.4% vs 4.5%; P = .02) but not with AR FMRD variants. CONCLUSIONS: The prevalence of HLRCC discovered incidentally on germline testing is similar to recent population carrier estimates, and this suggests that this is a relatively common cancer syndrome. Compared with those with negative genetic testing, those with VUSs had an elevated risk of RCC, whereas those with AR FMRD variants did not.

Prioritizing genes for systematic variant effect mapping.

MOTIVATION: When rare missense variants are clinically interpreted as to their pathogenicity, most are classified as variants of uncertain significance (VUS). Although functional assays can provide strong evidence for variant classification, such results are generally unavailable. Multiplexed assays of variant effect can generate experimental 'variant effect maps' that score nearly all possible missense variants in selected protein targets for their impact on protein function. However, these efforts have not always prioritized proteins for which variant effect maps would have the greatest impact on clinical variant interpretation. RESULTS: Here, we mined databases of clinically interpreted variants and applied three strategies, each building on the previous, to prioritize genes for systematic functional testing of missense variation. The strategies ranked genes (i) by the number of unique missense VUS that had been reported to ClinVar; (ii) by movability- and reappearance-weighted impact scores, to give extra weight to reappearing, movable VUS and (iii) by difficulty-adjusted impact scores, to account for the more resource-intensive nature of generating variant effect maps for longer genes. Our results could be used to guide systematic functional testing of missense variation toward greater impact on clinical variant interpretation. AVAILABILITY AND IMPLEMENTATION: Source code available at: https://github.com/rothlab/mave-gene-prioritization. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Systematic use of phenotype evidence in clinical genetic testing reduces the frequency of variants of uncertain significance.

Guidelines for variant interpretation include criteria for incorporating phenotype evidence, but this evidence is inconsistently applied. Systematic approaches to using phenotype evidence are needed. We developed a method for curating disease phenotypes as highly or moderately predictive of variant pathogenicity based on the frequency of their association with disease-causing variants. To evaluate this method's accuracy, we retrospectively reviewed variants with clinical classifications that had evolved from uncertain to definitive in genes associated with curated predictive phenotypes. To demonstrate the clinical validity and utility of this approach, we compared variant classifications determined with and without predictive phenotype evidence. The curation method was accurate for 93%-98% of eligible variants. Among variants interpreted using highly predictive phenotype evidence, the percentage classified as pathogenic or likely pathogenic was 80%, compared with 46%-54% had the evidence not been used. Positive results among individuals harboring variants with highly predictive phenotype-guided interpretations would have been missed in 25%-37% of diagnostic tests and 39%-50% of carrier screens had other approaches to phenotype evidence been used. In summary, predictive phenotype evidence associated with specific curated genes can be systematically incorporated into variant interpretation to reduce uncertainty and increase the clinical utility of genetic testing.

Elucidating clinical phenotypic variability associated with the polyT tract and TG repeats in CFTR.

Biallelic pathogenic variants in CFTR manifest as cystic fibrosis (CF) or other CFTR-related disorders (CFTR-RDs). The 5T allele, causing alternative splicing and reduced protein activity, is modulated by the adjacent TG repeat element, though previous data have been limited to small, selective cohorts. Here, the risk and spectrum of phenotypes associated with the CFTR TG-T5 haplotype variants (TG11T5, TG12T5, and TG13T5) in the absence of the p.Arg117His variant are evaluated. Individuals who received physician-ordered next-generation sequencing of CFTR were included. TG[11-13]T5 variant frequencies (biallelic or with another CF-causing variant [CFvar]) were calculated. Clinical information reported by the ordering provider or the individual was examined. Among 548,300 individuals, the T5 minor allele frequency (MAF) was 4.2% (TG repeat distribution: TG11 = 68.1%, TG12 = 29.5%, TG13 = 2.4%). When present with a CFvar, each TG[11-13]T5 variant was significantly enriched in individuals with a high suspicion of CF or CFTR-RD (personal/family history of CF/CFTR-RD) compared to those with a low suspicion for CF or CFTR-RD (hereditary cancer screening, CFTR not requisitioned). Compared to CFvar/CFvar individuals, those with TG[11-13]T5/CFvar generally had single-organ involvement, milder symptoms, variable expressivity, and reduced penetrance. These data improve our understanding of disease risks associated with TG[11-13]T5 variants and have important implications for reproductive genetic counseling.

Correction: Sherloc: a comprehensive refinement of the ACMG-AMP variant classification criteria.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Primary Ciliary Dyskinesia: Longitudinal Study of Lung Disease by Ultrastructure Defect and Genotype.

RATIONALE: In primary ciliary dyskinesia, factors leading to disease heterogeneity are poorly understood. OBJECTIVES: To describe early lung disease progression in primary ciliary dyskinesia and identify associations between ultrastructural defects and genotypes with clinical phenotype. METHODS: This was a prospective, longitudinal (5 yr), multicenter, observational study. Inclusion criteria were less than 19 years at enrollment and greater than or equal to two annual study visits. Linear mixed effects models including random slope and random intercept were used to evaluate longitudinal associations between the ciliary defect group (or genotype group) and clinical features (percent predicted FEV1 and weight and height z-scores). MEASUREMENTS AND MAIN RESULTS: A total of 137 participants completed 732 visits. The group with absent inner dynein arm, central apparatus defects, and microtubular disorganization (IDA/CA/MTD) (n = 41) were significantly younger at diagnosis and in mixed effects models had significantly lower percent predicted FEV1 and weight and height z-scores than the isolated outer dynein arm defect (n = 55) group. Participants with CCDC39 or CCDC40 mutations (n = 34) had lower percent predicted FEV1 and weight and height z-scores than those with DNAH5 mutations (n = 36). For the entire cohort, percent predicted FEV1 decline was heterogeneous with a mean (SE) decline of 0.57 (0.25) percent predicted/yr. Rate of decline was different from zero only in the IDA/MTD/CA group (mean [SE], -1.11 [0.48] percent predicted/yr; P = 0.02). CONCLUSIONS: Participants with IDA/MTD/CA defects, which included individuals with CCDC39 or CCDC40 mutations, had worse lung function and growth indices compared with those with outer dynein arm defects and DNAH5 mutations, respectively. The only group with a significant lung function decline over time were participants with IDA/MTD/CA defects.

Nasal Nitric Oxide in Primary Immunodeficiency and Primary Ciliary Dyskinesia: Helping to Distinguish Between Clinically Similar Diseases.

PURPOSE: Primary ciliary dyskinesia (PCD) is a rare disorder of the mucociliary clearance leading to recurrent upper and lower respiratory tract infections. PCD is difficult to clinically distinguish from other entities leading to recurrent oto-sino-pulmonary infections, including primary immunodeficiency (PID). Nasal nitric oxide (nNO) is a sensitive and specific diagnostic test for PCD, but it has not been thoroughly examined in PID. Past publications have suggested an overlap in nNO levels among subjects with PCD and PID. We sought to determine if nNO measurements among patients diagnosed with PID would fall significantly above the established PCD diagnostic cutoff value of 77 nL/min. METHODS: Children > 5 years old and adults with definitive PID or PCD diagnoses were recruited from outpatient subspecialty clinics. Participants underwent nNO testing by standardized protocol using a chemiluminescence analyzer and completed a questionnaire concerning their chronic oto-sino-pulmonary symptoms, including key clinical criteria specific to diagnosed PCD (neonatal respiratory distress at term birth, year-round cough or nasal congestion starting before 6 months of age, any organ laterality defect). RESULTS: Participants included 32 patients with PID, 27 patients with PCD, and 19 healthy controls. Median nNO was 228.9.1 nL/min in the PID group, 19.7 nL/min in the PCD group, and 269.4 in the healthy controls (p < 0.0001). Subjects with PCD were significantly more likely to report key clinical criteria specific to PCD, but approximately 25% of PID subjects also reported at least 1 of these key clinical criteria (mainly year-round cough or nasal congestion). CONCLUSIONS: While key clinical criteria associated with PCD often overlap with the symptoms reported in PID, nNO measurement by chemiluminescence technology allows for effective discrimination between PID and PCD.

Frequency of Cystic Fibrosis Transmembrane Conductance Regulator Variants in Individuals Evaluated for Primary Ciliary Dyskinesia.

OBJECTIVE: To evaluate whether cystic fibrosis transmembrane conductance regulator (CFTR) variants are more common among individuals tested for primary ciliary dyskinesia (PCD) compared with controls. STUDY DESIGN: Data were studied from 1021 individuals with commercial genetic testing for suspected PCD and 91 777 controls with genetic testing at the same company (Invitae) for symptoms/diseases unrelated to PCD or CFTR testing. The prevalence of CFTR variants was compared between controls and each of 3 groups of individuals tested for PCD (PCD-positive, -uncertain, and -negative molecular diagnosis). RESULTS: The prevalence of 1 pathogenic CFTR variant was similar among the individual groups. When combining the PCD-uncertain and PCR-negative molecular diagnosis groups, there was a higher prevalence of single pathogenic CFTR variants compared with controls (P = .03). Importantly, >1% of individuals who had negative genetic testing results for PCD had 2 pathogenic CFTR variants (8 of 723), and the incidence of cystic fibrosis (CF) (2 pathogenic variants) is roughly 1 in 3000 individuals of Caucasian ethnicity (∼0.03%). This incidence was also greater than that of 2 pathogenic CFTR variants in the control population (0.09% [84 of 91 777]; P = 9.60 × 10-16). These variants correlate with mild CFTR-related disease. CONCLUSIONS: Our results suggest that a single pathogenic CFTR variant is not likely to be a PCD-mimetic, but ongoing studies are needed in individuals in whom PCD is suspected and genetic testing results are uncertain or negative. Furthermore, CF may be misdiagnosed as PCD, reflecting phenotypic overlap. Among individuals evaluated for PCD, CF should be considered in the differential even in the CF newborn screening era.

Corrigendum: Sources of discordance among germ-line variant classifications in ClinVar.

This corrects the article DOI: 10.1038/gim.2017.60.

Sources of discordance among germ-line variant classifications in ClinVar.

PurposeClinVar is increasingly used as a resource for both genetic variant interpretation and clinical practice. However, controversies exist regarding the consistency of classifications in ClinVar, and questions remain about how best to use these data. Our study systematically examined ClinVar to identify common sources of discordance and thus inform ongoing practices.MethodsWe analyzed variants that had multiple classifications in ClinVar, excluding benign polymorphisms. Classifications were categorized by potential actionability and pathogenicity. Consensus interpretations were calculated for each variant, and the properties of the discordant outlier classifications were summarized.ResultsOur study included 74,065 classifications of 27,224 unique variants in 1,713 genes. We found that (i) concordance rates differed among clinical areas and variant types; (ii) clinical testing methods had much higher concordance than basic literature curation and research efforts; (iii) older classifications had greater discordance than newer ones; and (iv) low-penetrance variants had particularly high discordance.ConclusionRecent variant classifications from clinical testing laboratories have high overall concordance in many (but not all) clinical areas. ClinVar can be a reliable resource supporting variant interpretation, quality assessment, and clinical practice when factors uncovered in this study are taken into account. Ongoing improvements to ClinVar may make it easier to use, particularly for nonexpert users.

Sherloc: a comprehensive refinement of the ACMG-AMP variant classification criteria.

PurposeThe 2015 American College of Medical Genetics and Genomics-Association for Molecular Pathology (ACMG-AMP) guidelines were a major step toward establishing a common framework for variant classification. In practice, however, several aspects of the guidelines lack specificity, are subject to varied interpretations, or fail to capture relevant aspects of clinical molecular genetics. A simple implementation of the guidelines in their current form is insufficient for consistent and comprehensive variant classification.MethodsWe undertook an iterative process of refining the ACMG-AMP guidelines. We used the guidelines to classify more than 40,000 clinically observed variants, assessed the outcome, and refined the classification criteria to capture exceptions and edge cases. During this process, the criteria evolved through eight major and minor revisions.ResultsOur implementation: (i) separated ambiguous ACMG-AMP criteria into a set of discrete but related rules with refined weights; (ii) grouped certain criteria to protect against the overcounting of conceptually related evidence; and (iii) replaced the "clinical criteria" style of the guidelines with additive, semiquantitative criteria.ConclusionSherloc builds on the strong framework of 33 rules established by the ACMG-AMP guidelines and introduces 108 detailed refinements, which support a more consistent and transparent approach to variant classification.

Autosomal recessive MFN2-related Charcot-Marie-Tooth disease with diaphragmatic weakness: Case report and literature review.

Pathogenic variants in the mitofusin 2 gene (MFN2) are the most common cause of autosomal dominant Charcot-Marie-Tooth (CMT2) disease, which is typically characterized by axonal sensorimotor neuropathy. We report on a 7-month-old white female with hypotonia, motor delay, distal weakness, and motor/sensory axonal neuropathy in which next-generation sequencing analysis identified compound heterozygous pathogenic variants (c.2054_2069_1170del and c.392A>G) in MFN2. A review of the literature reveals that sporadic and familial cases of compound heterozygous or homozygous pathogenic MFN2 variants have been infrequently described, which indicates that MFN2 can also be inherited in a recessive manner. This case highlights several clinical findings not typically associated with MFN2 pathogenic variants, including young age of onset and rapidly progressing diaphragmatic paresis that necessitated tracheostomy and mechanical ventilation, and adds to the growing list of features identified in autosomal recessive MFN2-related CMT2. Our patient with MFN2-related CMT2 expands the clinical and mutational spectrum of individuals with autosomal recessive CMT2 and identifies a new clinical feature that warrants further observation. © 2016 Wiley Periodicals, Inc.

Expanding CXCR4 variant landscape in WHIM syndrome: integrating clinical and functional data for variant interpretation.

Warts, Hypogammaglobulinemia, Infections, Myelokathexis (WHIM) syndrome is a rare, combined immunodeficiency disease predominantly caused by gain-of-function variants in the CXCR4 gene that typically results in truncation of the carboxyl terminus of C-X-C chemokine receptor type 4 (CXCR4) leading to impaired leukocyte egress from bone marrow to peripheral blood. Diagnosis of WHIM syndrome continues to be challenging and is often made through clinical observations and/or genetic testing. Detection of a pathogenic CXCR4 variant in an affected individual supports the diagnosis of WHIM syndrome but relies on an appropriate annotation of disease-causing variants. Understanding the genotypic-phenotypic associations in WHIM syndrome has the potential to improve time to diagnosis and guide appropriate clinical management, resulting in a true example of precision medicine. This article provides an overview of the spectrum of CXCR4 variants in WHIM syndrome and summarizes the various lines of clinical and functional evidence that can support interpretation of newly identified variants.

Heterozygous loss-of-function SMC3 variants are associated with variable growth and developmental features.

Heterozygous missense variants and in-frame indels in SMC3 are a cause of Cornelia de Lange syndrome (CdLS), marked by intellectual disability, growth deficiency, and dysmorphism, via an apparent dominant-negative mechanism. However, the spectrum of manifestations associated with SMC3 loss-of-function variants has not been reported, leading to hypotheses of alternative phenotypes or even developmental lethality. We used matchmaking servers, patient registries, and other resources to identify individuals with heterozygous, predicted loss-of-function (pLoF) variants in SMC3, and analyzed population databases to characterize mutational intolerance in this gene. Here, we show that SMC3 behaves as an archetypal haploinsufficient gene: it is highly constrained against pLoF variants, strongly depleted for missense variants, and pLoF variants are associated with a range of developmental phenotypes. Among 14 individuals with SMC3 pLoF variants, phenotypes were variable but coalesced on low growth parameters, developmental delay/intellectual disability, and dysmorphism, reminiscent of atypical CdLS. Comparisons to individuals with SMC3 missense/in-frame indel variants demonstrated an overall milder presentation in pLoF carriers. Furthermore, several individuals harboring pLoF variants in SMC3 were nonpenetrant for growth, developmental, and/or dysmorphic features, and some had alternative symptomatologies with rational biological links to SMC3. Analyses of tumor and model system transcriptomic data and epigenetic data in a subset of cases suggest that SMC3 pLoF variants reduce SMC3 expression but do not strongly support clustering with functional genomic signatures of typical CdLS. Our finding of substantial population-scale LoF intolerance in concert with variable growth and developmental features in subjects with SMC3 pLoF variants expands the scope of cohesinopathies, informs on their allelic architecture, and suggests the existence of additional clearly LoF-constrained genes whose disease links will be confirmed only by multilayered genomic data paired with careful phenotyping.

Broadening the ciliopathy spectrum: motile cilia dyskinesia, and nephronophthisis associated with a previously unreported homozygous mutation in the INVS/NPHP2 gene.

Nephronophthisis associated ciliopathies (NPHP-AC) are a group of phenotypically related conditions that include Joubert syndrome, Meckel syndrome, nephronophthisis (NPHP), and Senior-Loken syndrome. We report on a male fetus with prenatal ultrasound findings at 24 weeks of gestation of anhydramnios, large and echogenic kidneys and situs inversus totalis. Histopathology revealed nephronophthisis and tracheal mucosa electron microscopy revealed ciliary dysgenesis. DNA analysis of the NPHP genes showed a previously unreported homozygous mutation, p.Arg603* (c.1078+1G>A), in the INVS/NPHP2 gene. This mutation is thought to abolish the splice donor site for exon 8, which likely disrupts the normal splicing of the INVS/NPHP2 gene.