Novel Pathogenic Mutation Mapping of ASPM Gene in Consanguineous Pakistani Families with Primary Microcephaly

Autosomal recessive primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by a congenitally reduced head circumference (-3 to -5 SD) and non-progressive intellectual disability. The objective of the study was to evaluate pathogenic mutations in the ASPM gene to understand etiology and molecular mechanism of primary microcephaly. Blood samples were collected from various families across different remote areas of Pakistan from February 2017 to May 2019 who were identified to be affected with primary microcephaly. DNA extraction was performed using the salting-out method; the quality and quantity of DNA were evaluated using spectrophotometry and 1% agarose gel electrophoresis, respectively in University of the Punjab. Mutation analysis was performed by whole exome sequencing from the Cologne Center for Genomics, University of Cologne. Sanger sequencing was done in University of the Punjab to confirm the pathogenic nature of mutation. A novel 4-bp deletion mutation c.3877_3880delGAGA was detected in exon 17 of the ASPM gene in two primary microcephaly affected families (A and B), which resulted in a frame shift mutation in the gene followed by truncated protein synthesis (p.Glu1293Lysfs*10), as well as the loss of the calmodulin-binding IQ domain and the Armadillo-like domain in the ASPM protein. Using the in-silico tools Mutation Taster, PROVEAN, and PolyPhen, the pathogenic effect of this novel mutation was tested; it was predicted to be “disease causing,” with high pathogenicity scores. One previously reported mutation in exon 24 (c.9730C>T) of the ASPM gene resulting in protein truncation (p.Arg3244*) was also observed in family C. Mutations in the ASPM gene are the most common cause of MCPH in most cases. Therefore, enrolling additional affected families from remote areas of Pakistan would help in identifying or mapping novel mutations in the ASPM gene of primary microcephaly.

Causal and Putative Pathogenic Mutations Identified in 38.6% of Children with Primary SRNS in South Africa

The aim was to identify causal mutations in genes implicated in steroid resistant nephrotic syndrome (SRNS) within a South African population. We enrolled 119 children with primary NS; 71 SRNS and 48 steroid-sensitive NS. All children with SRNS underwent kidney biopsy. We first genotyped the NPHS2 gene for the p.V260E variant in all NS cases (n= 119) and controls (n= 219). To further identify additional variants, we performed whole-exome sequencing and interrogated ten genes (NPHS1, NPHS2, WT1, LAMB2, ACTN4, TRPC6, INF2, CD2AP, PLCE1, MYO1E) implicated in SRNS/FSGS in 56 SRNS cases and 29 controls; we also performed exome sequencing on two patients carrying the NPHS2 p.V260E mutation as positive controls. The overall detection rate of pathogenic mutations in children with SRNS was 27/70(38.57%): 15(21.43%) carried the NPHS2 p.V260E mutation and 12(17.14%) carried a pathogenic mutation in the heterozygous state in INF2 (n=8), CD2AP (n=3) or TRPC6 (n=1) genes. NPHS2 p.V260E homozygosity was specifically associated with biopsy-proven FSGS, accounting for 23.81% of Black children (15 of 63) with SR-FSGS. No causal mutations were identified in NPHS1, WT1, LAMB2, PLCE1, MYO1E and ACTN4. We report four novel variants in INF2, PLCE1, ACTN4 and TRPC6.Conclusion: The NPHS2 p.V260E mutation is a prevalent cause of SR-FSGS among Black South African children occurring in 23.81% of children with SRNS. Screening all Black African children presenting with NS for NPHS2 p.V260E will provide a precision diagnosis of SR-FSGS and inform clinical management.

Successful Results of Intracytoplasmic Sperm Injection of a Chinese Patient With Multiple Morphological Abnormalities of Sperm Flagella Caused by a Novel Splicing Mutation in CFAP251

Asthenospermia is one of the most important causes of male infertility. Among asthenospermia, multiple morphological abnormalities of sperm flagella (MMAF) are relatively rare idiopathic conditions characterized by multiple defects in sperm flagella. Although many studies focusing on the genetic factors of MMAF have been conducted, its pathogenesis and treatment effect remain largely unknown. Here, we report a male patient from a nonconsanguineous Chinese family who exhibited a typical MMAF phenotype revealed by morphological analysis. We identified splicing mutations in CFAP251 (c.1192-3C>G), and the mutation was proven to cause exon skipping. In addition, western blotting and immunofluorescence analysis of the spermatozoa from the proband and a control subject revealed a significantly lower expression of CFAP251 protein due to pathogenic mutation. Interestingly, the patient’s mother was a heterozygous carrier for the mutation, but his father was not, and finally, the inheritance pattern was proven to be maternal uniparental disomy. We applied an intracytoplasmic sperm injection and achieved a successful pregnancy. Above all, our findings expand the spectrum of CFAP251 pathogenic mutations and provide more indications for clinical genetic counseling and assisted reproductive treatment for such patients.

A Three-Generation Muckle-Wells Syndrome Family: Detailed Family History, Physical Examination, and Inter-Departmental Collaboration

Cryopyrin-associated periodic syndrome (CAPS) is a rare inherited autoinflammatory disease caused by gain-of-function mutations in the NLRP3 gene, with a genotype-phenotype correlation. The clinical presentation of each mutation has been previously studied. However, very few studies have reported on the clinical characteristics and treatment effectiveness across different generations within a family with the same mutation. A detailed investigation of family members of patients with CAPS may help in the appropriate diagnosis and treatment of undiagnosed CAPS. Herein, we report a 2-year-old boy (proband), his father, and his grandmother who presented with several symptoms of CAPS, such as persistently positive inflammatory reactions and hearing impairment. All three patients had the same pathogenic mutation in the NLRP3 gene (c.1049C>T (p.Thr350Met) heterozygous mutation) and were diagnosed with CAPS. With canakinumab treatment, the laboratory data of all three patients improved, the proband and father’s skin rash disappeared, and his grandmother’s arthropathy improved. The proband’s hearing also showed slight improvement, but not in his father or grandmother. Among the various non-specific symptoms associated with CAPS, chronic ocular hyperemia is a finding that can be easily identified by non-ophthalmologists. Diagnosis of CAPS should be considered when eye symptoms are present in a combination of hyperinflammatory response, arthropathy, or skin symptoms. Thorough family history records, physical examinations, and close collaboration between pediatricians and adult rheumatologists are important for prompt diagnosis and appropriate treatment of inherited autoinflammatory diseases.

Prime Editor-mediated Correction of a Pathogenic Mutation in Purebred Dogs

Canine hip dysplasia (HD) is a multifactorial disease caused by interactions between genetic and environmental factors. HD, which mainly occurs in medium- to large-sized dogs, is a disease that causes severe pain and requires surgical intervention. However, the procedure is not straight-forward, and the only way to ameliorate the situation is to exclude individual dogs with HD from breeding programs. Recently, prime editing (PE), a novel genome editing tool based on the CRISPR-Cas9 system, has been developed and validated in plants and mice. In this study, we successfully corrected a mutation related to HD in Labrador retriever dogs for the first time. We collected cells from a dog diagnosed with HD, corrected the mutation using PE, and generated mutation-corrected dogs by somatic cell nuclear transfer. The results indicate that PE technology can potentially be used as a platform to correct genetic defects in dogs.

Reviewing the Clinical Spectrum Related to Kmt2b Gene Mutations: Unusual Clinical Presentation and a Possible New Pathogenic Mutation

Introduction KMT2B related dystonia is a childhood onset generalized dystonia. Since its first description in 2016, different phenotypic spectrum have been reported. The aim of the case report is to provide data that may help to understand the spectrum of KMT2B-related disorders. We present two members of a family with a possible non-previously described pathogenic mutation and an unusual KMT2B related dystonia presentation: an adult onset and focal dystonia.Case Presentation The index patient is a 32 year-old woman with a generalized dystonia. Her maternal uncle presented a focal dystonia. Next-generation sequencing revealed a heterozygous missense mutation in KMT2B gene (19q13.12), described as a variant of uncertain significance (VUS). Although characteristic phenotype of KMT2B dystonia is a childhood onset generalized dystonia, different phenotypes have been related according to the kinds of mutations in this gene, also varying the age of symptom onset and the penetrance of the mutation. Asymptomatic or sub-clinical carriers and adult onset has been described. Due to the low prevalence of this variant in the general population and the low penetrance and high intrafamilial variability of this entity, we suggest that this mutation might be a pathogenic variant.ConclusionsKMT2B related dystonia is an emerging and prevalent monogenic dystonia whose incidence, genetic variability and clinical spectrum remain unknown. Despite the study of this gene is indicated in childhood onset dystonia, description of cases such as ours shows that its sequencing in patients with an adult-onset dystonia with family history can be useful for the diagnosis.

Giant Gastric Folds in Juvenile Polyposis

To advance the diagnostic accuracy of juvenile polyposis syndrome, an important yet often difficult diagnosis, we describe in detail a new and medically significant presentation. This hereditary and high-risk GI cancer syndrome is often associated with hereditary hemorrhagic telangiectasia, as in this 47-year-old female patient with a <i>SMAD4</i> germline pathogenic mutation. Total gastrectomy revealed giant gastric folds with inflamed foveolar hyperplasia consuming most of the gastric cardia and body but sparing the antrum. Together, this gross and histologic pathology mimics Ménétrier’s disease, an exceedingly rare and acquired protein-losing hypertrophic gastropathy. Classical gastric juvenile polyposis almost always and principally involves the antrum with multiple distinctive inflammatory <i>polyps</i> rather than the newly illustrated <i>giant gastric folds</i> of this case. No reports of giant gastric folds in juvenile polyposis have appeared in the literature. The distinction between juvenile polyposis and Ménétrier’s disease is essential due to their disparate clinical outcomes and management. The differential considerations for giant gastric folds and inflamed gastric foveolar hyperplasia are fully reviewed. On the basis of this report, the differential for giant gastric folds must now expand to include juvenile polyposis syndrome. Genetic testing for pathogenic germline mutations of the 2 known causative genes of this syndrome, namely <i>SMAD4</i> and <i>BMPR1A</i>, are readily available and should become part of the evaluation of giant gastric folds, particularly in view of the neoplastic and hereditary aspects of juvenile polyposis syndrome.

Expression of Programmed Cell Death-L1 (PD-L1) Protein and Mismatch Repair Mutations in Orbital Tumours-a Pilot Study

Purpose Programmed cell death protein 1 (PD-1) and DNA mismatch repair (MMR) deficiency play an important role in tumour progression and response to treatment. Both markers have been studied in some ocular tumours but little is known about these markers in orbital tumours. This pilot study reports on PD-L1 expression and MMR mutations using next generation sequencing (NGS) in specific orbital tumours. Methods We reviewed surgical specimens from patients with rhabdomyosarcoma, adenoid cystic carcinoma (ACC), pleomorphic adenoma (PA) and biopsy negative tissue from orbital tumours used as a control. immunohistochemistry (IHC) was performed on Formalin fixed paraffin embedded tissue using a PD-L1 antibody. DNA was extracted for targeted gene panel NGS of the MMR genes PMS2, MLH1, MSH6 and MSH2. Results The study included 17 orbital specimens. Scattered membrane PD-L1 staining was noted in 3/6 rhabdomyosarcoma specimens without an accompanying lymphocytic infiltrate. PD-L1 immunostaining was absent in 3/3 ACC, and 5/6 PA specimens. PD-L1 immunostaining was not detected in 2/2 control specimens. 4/17 samples shared the same pathogenic mutation in the MLH1 gene, including 3/6 rhabdomyosarcoma and 1/3 ACC samples. 1/6 PA samples had a mutation in MSH6. Conclusions Our study demonstrated scattered, non-quantifiable or absent PD-L1 staining in a limited sample of orbital tumours suggesting that PD-1/PD-L1 inhibitor therapy may not be useful in treatment of malignant orbital tumours (rhabdomyosarcoma and ACC) when refractory to conventional therapy. Our pilot study suggest that PD-L1/MMR axis might not play a major role in the pathogenesis of primary orbital tumour.