Case report: a novel deep intronic splice-altering variant in DMD as a cause of Becker muscular dystrophy

We present the case of a male patient who was ultimately diagnosed with Becker muscular dystrophy (BMD; MIM# 300376) after the onset of muscle weakness in his teens progressively led to significant walking difficulties in his twenties. A genetic diagnosis was pursued but initial investigation revealed no aberrations in the dystrophin gene (DMD), although immunohistochemistry and Western blot analysis suggested the diagnosis of dystrophinopathy. Eventually, after more than 10 years, an RNA analysis captured abnormal splicing where 154 nucleotides from intron 43 were inserted between exon 43 and 44 resulting in a frameshift and a premature stop codon. Normal splicing of the DMD gene was also observed. Additionally, a novel variant c.6291–13537A>G in DMD was confirmed in the genomic DNA of the patient. The predicted function of the variant aligns with the mRNA results. To conclude, we here demonstrate that mRNA analysis can guide the diagnosis of non-coding genetic variants in DMD

Clinical and Genetic Studies of Hearing Impairment

Monogenic disorders offer a possibility for studies of genetic disturbances in hearing impairment—a knowledge which could be essential for development of future treatment options. In this thesis, the underlying genetic disturbances in neurofibromatosis 2 (NF2) and familial Meniere’s disease (FMD) were evaluated, and familial X-linked hearing impairment was described from a clinical point of view. In paper I, constitutional DNA from 116 individuals with NF2 of variable severity was studied using the array-CGH method focusing on a 7.6-Mb area surrounding the NF2 gene on chromosome 22q. Deletions were found in 20.7% of samples. In mild NF2, the deletions were small, but variable sizes of deletions were found in cases that were moderately or severely affected. Disease phenotype could not be predicted from the size of the deletions. In papers II and III, a single five-generation family with autosomal dominant FMD was described. Anticipation concerning age of onset was observed. Genome scan revealed five candidate gene regions with a LOD score of > 1. Two additional families with autosomal dominant MD were analyzed for linkage to these five regions. A cumulative Zmax of 3.46 was obtained for a single 463-kb region on chromosome 12p12.3, containing only one known gene: PIK3C2G. This encodes a protein with a proposed role in hair cell regeneration in mammalian ears. No mutations were found in protein-coding sequences or exon-intron borders. In two of the three families, a shared haplotype, suggested common ancestry, was found to extend over 1.7 Mb, which could be a genomic region of importance for FMD. In paper IV, a family in which five males displayed progressive low- and mid-frequency hearing impairment from the first or second decade was described. Female carriers were affected by a high-frequency hearing impairment from the fourth decade. The family could represent a novel X-linked dominant audiophenotype

Clinical and Genetic Studies of Hearing Impairment

Monogenic disorders offer a possibility for studies of genetic disturbances in hearing impairment—a knowledge which could be essential for development of future treatment options. In this thesis, the underlying genetic disturbances in neurofibromatosis 2 (NF2) and familial Meniere’s disease (FMD) were evaluated, and familial X-linked hearing impairment was described from a clinical point of view. In paper I, constitutional DNA from 116 individuals with NF2 of variable severity was studied using the array-CGH method focusing on a 7.6-Mb area surrounding the NF2 gene on chromosome 22q. Deletions were found in 20.7% of samples. In mild NF2, the deletions were small, but variable sizes of deletions were found in cases that were moderately or severely affected. Disease phenotype could not be predicted from the size of the deletions. In papers II and III, a single five-generation family with autosomal dominant FMD was described. Anticipation concerning age of onset was observed. Genome scan revealed five candidate gene regions with a LOD score of > 1. Two additional families with autosomal dominant MD were analyzed for linkage to these five regions. A cumulative Zmax of 3.46 was obtained for a single 463-kb region on chromosome 12p12.3, containing only one known gene: PIK3C2G. This encodes a protein with a proposed role in hair cell regeneration in mammalian ears. No mutations were found in protein-coding sequences or exon-intron borders. In two of the three families, a shared haplotype, suggested common ancestry, was found to extend over 1.7 Mb, which could be a genomic region of importance for FMD. In paper IV, a family in which five males displayed progressive low- and mid-frequency hearing impairment from the first or second decade was described. Female carriers were affected by a high-frequency hearing impairment from the fourth decade. The family could represent a novel X-linked dominant audiophenotype

Novel pathogenic ALG2 mutation causing congenital myasthenic syndrome : A case report

ALG2 mutations are extremely rare causes of congenital myasthenic syndromes (CMS). The clinical phenotype and treatment response is therefore not well described. We present the case of a baby who immediately after birth presented with pronounced truncal hypotonia, proximal muscle weakness and feeding difficulties. Single fibre electromyography showed neuromuscular transmission failure and salbutamol and ephedrine treatment improved both muscle weakness and neuromuscular transmission. Genetic analysis revealed a likely pathogenic variant c.1040del, p.(Gly347Valfs*27) in exon 2 and a variant of uncertain significance, c.239G>A, p.(Gly80Asp) in exon 1 of the ALG2 gene. Western blot in whole cell lysates of HEK293 cells transfected with p.Gly80Asp, or p.Gly347Valfs*27 expression constructs indicated that p.Gly347Valfs*27 is likely a null allele and p.Gly80Asp is pathogenic through marked reduction of ALG2 expression. This case highlights the utility of functional studies in clarifying variants of unknown significance, in suspected cases of CMS. (C) 2021 The Author(s). Published by Elsevier B.V

A novel approach using long-read sequencing and ddPCR to investigate gonadal mosaicism and estimate recurrence risk in two families with developmental disorders

Objective De novo mutations contribute significantly to severe early-onset genetic disorders. Even if the mutation is apparently de novo, there is a recurrence risk due to parental germ line mosaicism, depending on in which gonadal generation the mutation occurred. Methods We demonstrate the power of using SMRT sequencing and ddPCR to determine parental origin and allele frequencies of de novo mutations in germ cells in two families whom had undergone assisted reproduction. Results In the first family, a TCOF1 variant c.3156C>T was identified in the proband with Treacher Collins syndrome. The variant affects splicing and was determined to be of paternal origin. It was present in <1% of the paternal germ cells, suggesting a very low recurrence risk. In the second family, the couple had undergone several unsuccessful pregnancies where a de novo mutation PTPN11 c.923A>C causing Noonan syndrome was identified. The variant was present in 40% of the paternal germ cells suggesting a high recurrence risk. Conclusions Our findings highlight a successful strategy to identify the parental origin of mutations and to investigate the recurrence risk in couples that have undergone assisted reproduction with an unknown donor or in couples with gonadal mosaicism that will undergo preimplantation genetic diagnosis

Exome sequencing reveals NAA15 and PUF60 as candidate genes associated with intellectual disability

Intellectual Disability (ID) is a clinically heterogeneous condition that affects 2-3% of population worldwide. In recent years, exome sequencing has been a successful strategy for studies of genetic causes of ID, providing a growing list of both candidate and validated ID genes. In this study, exome sequencing was performed on 28 ID patients in 27 patient-parent trios with the aim to identify de novo variants (DNVs) in known and novel ID associated genes. We report the identification of 25 DNVs out of which five were classified as pathogenic or likely pathogenic. Among these, a two base pair deletion was identified in the PUF60 gene, which is one of three genes in the critical region of the 8q24.3 microdeletion syndrome (Verheij syndrome). Our result adds to the growing evidence that PUF60 is responsible for the majority of the symptoms reported for carriers of a microdeletion across this region. We also report variants in several genes previously not associated with ID, including a de novo missense variant in NAA15. We highlight NAA15 as a novel candidate ID gene based on the vital role of NAA15 in the generation and differentiation of neurons in neonatal brain, the fact that the gene is highly intolerant to loss of function and coding variation, and previously reported DNVs in neurodevelopmental disorders

Loss of Nexilin function leads to a recessive lethal fetal cardiomyopathy characterized by cardiomegaly and endocardial fibroelastosis

The Nexilin F-Actin Binding Protein (Nexilin) encoded by NEXN is a cardiac Z-disc protein important for cardiac function and development in humans, zebrafish, and mice. Heterozygote variants in the human NEXN gene have been reported to cause dilated and hypertrophic cardiomyopathy. Homozygous variants in NEXN cause a lethal form of human fetal cardiomyopathy, only described in two patients before. In a Swedish, four-generation, non-consanguineous family comprising 42 individuals, one female had three consecutive pregnancies with intrauterine fetal deaths caused by a lethal form of dilated cardiomyopathy. Whole-exome sequencing and variant analysis revealed that the affected fetuses were homozygous for a NEXN variant (NM_144573:c.1302del;p.(Ile435Serfs*3)). Moreover, autopsy and histology staining declared that they presented with cardiomegaly and endocardial fibroelastosis. Immunohistochemistry staining for Nexilin in the affected fetuses revealed reduced antibody staining and loss of striation in the heart, supporting loss of Nexilin function. Clinical examination of seven heterozygote carriers confirmed dilated cardiomyopathy (two individuals), other cardiac findings (three individuals), or no cardiac deviations (two individuals), indicating incomplete penetrance or age-dependent expression of dilated cardiomyopathy. RNA sequencing spanning the variant in cDNA blood of heterozygote individuals revealed nonsense-mediated mRNA decay of the mutated transcripts. In the current study, we present the first natural course of the recessively inherited lethal form of human fetal cardiomyopathy caused by loss of Nexilin function. The affected family had uneventful pregnancies until week 23-24, followed by fetal death at week 24-30, characterized by cardiomegaly and endocardial fibroelastosis

Case report : a novel deep intronic splice-altering variant in DMD as a cause of Becker muscular dystrophy

We present the case of a male patient who was ultimately diagnosed with Becker muscular dystrophy (BMD; MIM# 300376) after the onset of muscle weakness in his teens progressively led to significant walking difficulties in his twenties. A genetic diagnosis was pursued but initial investigation revealed no aberrations in the dystrophin gene (DMD), although immunohistochemistry and Western blot analysis suggested the diagnosis of dystrophinopathy. Eventually, after more than 10 years, an RNA analysis captured abnormal splicing where 154 nucleotides from intron 43 were inserted between exon 43 and 44 resulting in a frameshift and a premature stop codon. Normal splicing of the DMD gene was also observed. Additionally, a novel variant c.6291–13537A>G in DMD was confirmed in the genomic DNA of the patient. The predicted function of the variant aligns with the mRNA results. To conclude, we here demonstrate that mRNA analysis can guide the diagnosis of non-coding genetic variants in DMD