Muhammad Imran Khan

Chronic pancreatitis (CP) is characterized by progressive fibrosis, inflammation, and pancreatic atrophy. Here, we evaluate a milder CP model using only 42 cerulein injections administered in both male and female C57BL/6J (BL6) and BALB/c mice. We further assess the therapeutic potential of the RORγt inhibitor GSK805. We employed a multi‐tiered analytical approach, including the robustness of conclusion (RC), unit fragility index, and robustness index, to evaluate consistency and numerical stability of conclusions. The model reliably produced key hallmarks of CP: reduced pancreas‐to‐body weight ratio, elevated collagen I deposition, and tubular complex formation with an RC of 100%. In contrast, the induction of Tnf , Ifng , Il10 , Il23r , Il6 , and Il1b expression was less robust and partially strain specific. Il23r and Il10 were significantly induced during CP in female and male BL6 but not in BALB/c mice. Il6 was significantly induced in female and male BALB/c but only reached significance in BL6 when sexes were combined. Treatment with GSK805 reduced Il23r expression level significantly in BL6 mice, but no reliable improvements in other disease characteristics were observed. Overall, this study validates a simplified, robust CP model and advocates for the integration of complementary statistical metrics to strengthen the reliability of scientific conclusion.

A substantial proportion of subjects with autosomal recessive retinitis pigmentosa (arRP) or Usher syndrome type II (USH2) lacks a genetic diagnosis due to incomplete USH2A screening in the early days of genetic testing. These cases lack eligibility for optimal genetic counseling and future therapy. USH2A defects are the most frequent cause of USH2 and are also causative in individuals with arRP. Therefore, USH2A is an important target for genetic screening. The aim of this study was to assess unscreened or incompletely screened and unexplained USH2 and arRP cases for (likely) pathogenic USH2A variants. Molecular inversion probe (MIP)-based sequencing was performed for the USH2A exons and their flanking regions, as well as published deep-intronic variants. This was done to identify single nucleotide variants (SNVs) and copy number variants (CNVs) in 29 unscreened or partially pre-screened USH2 and 11 partially pre-screened arRP subjects. In 29 out of these 40 cases, two (likely) pathogenic variants were successfully identified. Four of the identified SNVs and one CNV were novel. One previously identified synonymous variant was demonstrated to affect pre-mRNA splicing. In conclusion, genetic diagnoses were obtained for a majority of cases, which confirms that MIP-based sequencing is an effective screening tool for USH2A. Seven unexplained cases were selected for future analysis with whole genome sequencing.

In this retrospective, longitudinal, observational cohort study, we investigated the phenotypic and genotypic features of retinitis pigmentosa associated with variants in the PDE6B gene. Patients underwent clinical examination and genetic testing at a single tertiary referral center, including best-corrected visual acuity (BCVA), kinetic visual field (VF), full-field electroretinography, full-field stimulus threshold, spectral domain optical coherence tomography, and fundus autofluorescence imaging. The genetic testing comprised candidate gene sequencing, inherited retinal disease gene panel sequencing, whole-genome sequencing, and testing for familial variants by Sanger sequencing. Twenty-four patients with mutations in PDE6B from 21 families were included in the study (mean age at the first visit: 32.1 ± 13.5 years). The majority of variants were putative splicing defects (8/23) and missense (7/23) mutations. Seventy-nine percent (38/48) of eyes had no visual acuity impairment at the first visit. Visual acuity impairment was mild in 4% (2/48), moderate in 13% (6/48), and severe in 4% (2/48). BCVA was symmetrical in the right and left eyes. The kinetic VF measurements were highly symmetrical in the right and left eyes, as was the horizontal ellipsoid zone (EZ) width. Regarding the genetic findings, 43% of the PDE6B variants found in our patients were novel. Thus, this study contributed substantially to the PDE6B mutation spectrum. The visual acuity impairment was mild in 83% of eyes, providing a window of opportunity for investigational new drugs. The EZ width was reduced in all patients and was highly symmetric between the eyes, making it a promising outcome measure. We expect these findings to have implications on the design of future PDE6B-related retinitis pigmentosa (RP) clinical trials.

Purpose: To study the disease course ofRPE65-associated inherited retinal degenerations (IRDs) as a function of the genotype, define a critical age for blindness, and identify potential modifiers. Methods: Forty-five patients with IRD from 33 families with biallelicRPE65mutations, 28 stemming from a genetic isolate. We collected retrospective data from medical charts. Coexisting variants in 108 IRD-associated genes were identified with Molecular Inversion Probe analysis. Results: Most patients were diagnosed within the first years of life. Daytime visual function ranged from near-normal to blindness in the first four decades and met WHO criteria for blindness for visual acuity and visual field in the fifth decade. p.(Thr368His) was the most common variant (54%). Intrafamilial variability and interfamilial variability in disease severity and progression were observed. Molecular Inversion Probe analysis confirmed allRPE65variants and identified one additional variant inLRATand one inEYSin two separate patients. Conclusion: All patients withRPE65-associated IRDs developed symptoms within the first year of life. Visual function in childhood and adolescence varied but deteriorated inevitably toward blindness after age 40. In this study, genotype was not predictive of clinical course. The variance in severity of disease could not be explained by double hits in other IRD genes.

Mutations in retina-specific ATP-binding cassette transporter 4 (ABCA4) are responsible for over 95% of cases of Stargardt disease (STGD), as well as a minor proportion of retinitis pigmentosa (RP) and cone-rod dystrophy cases (CRD). Since the knowledge of the genetic causes of inherited retinal diseases (IRDs) in Poland is still scarce, the purpose of this study was to identify pathogenic ABCA4 variants in a subgroup of Polish IRD patients. We recruited 67 families with IRDs as a part of a larger study. The patients were screened with next generation sequencing using a molecular inversion probes (MIPs)-based technique targeting 108 genes involved in the pathogenesis of IRDs. All identified mutations were validated and their familial segregation was tested using Sanger sequencing. In the case of the most frequent complex allele, consisting of two variants in exon 12 and 21, familial segregation was tested using restriction fragment length polymorphism (RFLP). The most prevalent variant, a complex change c.[1622T>C;3113C>T], p.[Leu541Pro;Ala1038Val], was found in this cohort in 54% of all solved ABCA4-associated disorder cases, which is the highest frequency reported thus far. Additionally, we identified nine families displaying a pseudo-dominant mode of inheritance, indicating a high frequency of pathogenic variants within this population.

Purpose: Mutations in the CRB1 gene cause early-onset retinal degeneration (EORD). Clinical disease progression markers, such as visual fields or electrophysiology, are not reliably measurable in most patients to follow the retinal function in patients with CRB1-mutations. Methods: Ten patients (five females, five males; age 22–56 years) with EORD caused by CRB1 mutations were examined in a cross-sectional manner using best corrected visual acuity (BCVA), perimetry, full-field and multifocal electroretinography, full-field stimulus threshold (FST), and pupillography to red and blue light. Disease duration was defined as the difference between the age at the first symptoms to the age at examination in years. Results: BCVA was quantifiable in six patients and ranged from light perception to 20/50. The visual field was measurable only in three patients who had the shortest disease duration. Full-field and multifocal electroretinography were not measurable in any patient. FST to blue and red light were measurable in all patients except the one with the longest disease duration; the thresholds ranged from −16.7 to 1.5 dB for red light and from −40.2 to 2.5 dB for blue light (0 dB = 0.01 cd.s/m²) and showed correlations with disease duration (r = 0.87 for blue, r = 0.65 for red, r = 0.8 for blue–red difference). The maximal relative pupil constriction amplitude (MRA) showed low or no correlations with disease duration (r = −0.55 for blue, r = −0.3 for red light); the blue–red difference in the post-illumination pupil responses (PIPR) showed no correlation with disease duration (r = −0.05). Compared to healthy eyes, the MRA to red and blue light was significantly decreased (P < 0.001) and the blue–red PIPR difference was significantly increased (P = 0.003). Conclusions: FST features a valid clinical marker in late-stage early-onset retinitis pigmentosa caused by CRB1 mutations correlating with disease duration. This indicates the potential as a progression marker of disease. The pupil responses to full-field chromatic stimuli show significant differences from the normal population: the remaining responses, although reduced, indicate a partially preserved inner retinal function despite severe photoreceptor dysfunction. Translational Relevance: The functional measurements presented in this study present a valid clinical progression marker in late-stage early onset retinitis pigmentosa caused by biallelic CRB1 mutations. Additionally, they can be used as outcome measures for safety and efficacy in clinical therapy trials.

Inherited retinal diseases (IRDs) cause visual loss due to dysfunction or progressive degeneration of photoreceptors. These diseases show marked phenotypic and genetic heterogeneity. The Israeli IRD consortium (IIRDC) was established in 2013 with the goal of performing clinical and genetic mapping of the majority of Israeli IRD patients. To date we recruited 2,420 families including 3,413 individuals with IRDs. Based on our estimation, these patients represent approximately 40% of Israeli IRD patients. To the best of our knowledge, this is by far the largest reported IRD cohort, and one of the first studies addressing genetic analysis of IRD patients on a nation‐wide scale. The most common inheritance pattern in our cohort is autosomal recessive (60% of families). The most common retinal phenotype is retinitis pigmentosa (43%), followed by Stargardt disease and cone/cone‐rod dystrophy. We identified the cause of disease in 56% of the families. Overall, 605 distinct mutations were identified, of which 12% represent prevalent founder mutations. The most frequently mutated genes were ABCA4, USH2A, FAM161A, CNGA3 and EYS. The results of this study have important implications for molecular diagnosis, genetic screening and counseling, as well as for development of new therapeutic strategies for retinal diseases.

Hereditary retinal degenerations (HRDs) are Mendelian diseases characterized by progressive blindness and caused by ultra-rare mutations. In a genomic screen of 331 unrelated Japanese patients, we identify a disruptive Alu insertion and a nonsense variant (p.Arg1933*) in the ciliary gene RP1, neither of which are rare alleles in Japan. p.Arg1933* is almost polymorphic (frequency = 0.6%, amongst 12,000 individuals), does not cause disease in homozygosis or heterozygosis, and yet is significantly enriched in HRD patients (frequency = 2.1%, i.e., a 3.5-fold enrichment; p-value = 9.2 × 10⁻⁵). Familial co-segregation and association analyses show that p.Arg1933* can act as a Mendelian mutation in trans with the Alu insertion, but might also associate with disease in combination with two alleles in the EYS gene in a non-Mendelian pattern of heredity. Our results suggest that rare conditions such as HRDs can be paradoxically determined by relatively common variants, following a quasi-Mendelian model linking monogenic and complex inheritance.

Purpose Stargardt disease (STGD1) is caused by biallelic mutations in ABCA4, but many patients are genetically unsolved due to insensitive mutation‐scanning methods. We aimed to develop a cost‐effective sequencing method for ABCA4 exons and regions carrying known causal deep‐intronic variants. Methods Fifty exons and 12 regions containing 14 deep‐intronic variants of ABCA4 were sequenced using double‐tiled single molecule Molecular Inversion Probe (smMIP)‐based next‐generation sequencing. DNAs of 16 STGD1 cases carrying 29 ABCA4 alleles and of four healthy persons were sequenced using 483 smMIPs. Thereafter, DNAs of 411 STGD1 cases with one or no ABCA4 variant were sequenced. The effect of novel noncoding variants on splicing was analyzed using in vitro splice assays. Results Thirty‐four ABCA4 variants previously identified in 16 STGD1 cases were reliably identified. In 155/411 probands (38%), two causal variants were identified. We identified 11 deep‐intronic variants present in 62 alleles. Two known and two new noncanonical splice site variants showed splice defects, and one novel deep‐intronic variant (c.4539+2065C>G) resulted in a 170‐nt mRNA pseudoexon insertion (p.[Arg1514Lysfs*35,=]). Conclusions smMIPs‐based sequence analysis of coding and selected noncoding regions of ABCA4 enabled cost‐effective mutation detection in STGD1 cases in previously unsolved cases.

Purpose: Brittle cornea syndrome (BCS) is a rare recessive disorder affecting connective tissues, most prominently in the eye. Pathogenic mutations causing BCS have been identified in PRDM5 and ZNF469 genes. This study investigates the genetic cause of BCS in a large, consanguineous Pakistani family with 4 affected and 3 unaffected individuals. Methods: The coding region and exon–intron splice junctions of PRDM5 and ZNF469 genes were amplified by polymerase chain reaction, and bidirectional Sanger sequencing was performed to find the pathogenic change responsible for causing the disease in the family. Results: A novel homozygous duplication c.9831dupC (p.Arg3278GlnfsX197) in the ZNF469 gene was identified, which was found to be co-segregating with the disease in the family. Conclusions: This is the first report of a ZNF469 homozygous mutation causing a BCS phenotype in a consanguineous Pakistani family. Our data extend the mutation spectrum of ZNF469 variants implicated in BCS.