Novel Splice-Site and Missense Variant of PNPLA6 in an Austrian Family Causing Spastic Paraplegia-39 with Cerebellar Oculomotor Disorder

Background The term hereditary spastic paraplegia comprises an ever-expanding array of neurological disorders with distinct aetiologies. Spastic paraplegia 39 is one of the many variants with additional features of other organs and neurological systems. We describe a large kindred with two hitherto undescribed mutations of PNPLA6 and a novel clinical phenotype as, in addition to spastic paraplegia, affected subjects suffered from a prominent cerebellar oculomotor dysfunction. Methods Three of five genetically tested family members of a large kindred were affected by spastic gait and cerebellar oculomotor dysfunction. Clinical, imaging, laboratory and electrophysiological data were analyzed. Genetic analysis was done using next-generation sequencing. Segregation analyses were performed by Sanger sequencing. To assess the pathogenicity of genetic variants on the encoded protein, in silico assessments were carried out. Results Two hitherto unknown sequence variants in the PNPLA6 gene, a splice-site variant c.1635+3G>T and a missense variant c.3401A>T, p.(Asp1134Val), were detected. Compound-heterozygous siblings presented with lower limb spasticity and a marked cerebellar oculomotor disorder accompanied by moderate hypogonadotropic hypogonadism in the female. A paternal uncle being homozygous for the splice-site variant of PNPLA6 presented with increased lower limb reflexes, an unstable gait and cerebellar oculomotor dysfunction. Treatment with 4-aminopyridin, a potassium channel blocker, lead to meaningful improvement of clinical symptoms. Conclusion PNPLA6 gene variants are associated with a broad phenotypic spectrum to which we add cerebellar oculomotor dysfunction. In our kindred, the full clinical manifestation only occurred in compound-heterozygous subjects indicating that biallelic pathogenic variants lead to more serious and earlier onset of symptoms. Our findings emphasize the role of PNPLA6 in different neurodegenerative disorders.

P.123 Severe DNM1 Encephalopathy with Dysmyelination due to Recurrent Splice Site Pathogenic Variant

Background: Patients with DNM1-encephalopathy almost exclusively have missense variants, mostly in the GTPase domain of DNM1. Delayed myelination has been reported in at least three patients with DNM1-encephalopathy, all with missense mutations in the DNM1 central domain. Only one DNM1 splice-site variant has previously been reported, and the authors questioned whether the variant accounted for all aspects of the patient’s phenotype. Methods: Case-Report. Results: Our patient had hypotonia and brief multifocal tonic seizures from age-1-month. He still has profound global developmental delay, daily seizures and microcephaly. MRI-Brain at age-21-months showed T2 hyperintensity in the bilateral periventricular and subcortical white matter; spectroscopy showed a questionable lactate peak and an elevated choline peak relative to N-acetylaspartate. Clinical gene-panel identified a heterozygous de novo pathogenic variant in intron 9 of DNM1 (c.1197-8G > A; IVS9- 8G>A). In-silico tools categorized this variant as deleterious secondary to a splicing defect. RT-PCR analysis on peripheral blood was unsuccessful as DNM1 expression is extremely low outside of the brain. Conclusions: Our patient carried the same DNM1 variant previously reported, indicating this is a recurrent pathogenic splice-site variant. The spectroscopic abnormalities suggest a possible element of demyelination in DNM1 variants of the central domain, though the mechanism remains unclear.

Clear evidence of LAMA5 gene biallelic truncating variants causing infantile nephrotic syndrome

Background: Pathogenic variants in single genes encoding podocyte-associated proteins have been implicated in about 30% of steroid resistant nephrotic syndrome (SRNS) patients in children. However, LAMA5 gene biallelic variants have been identified in only 7 patients so far, and most are missense variants of unknown significance. Furthermore, no functional analysis had been conducted for all but one of these variants. Here, we report three patients with LAMA5 gene biallelic truncating variants manifesting infantile nephrotic syndrome and one SRNS case with biallelic LAMA5 missense variants. Methods: We conducted comprehensive gene screening of Japanese patients with severe proteinuria. Using targeted next-generation sequencing, 62 podocyte-related genes were screened in 407 unrelated patients with proteinuria. For the newly discovered LAMA5 variants, we conducted in vitro heterotrimer formation assays. Results: Biallelic truncating variants in the LAMA5 gene (NM_005560) were detected in 3 patients from 2 families. All patients presented with proteinuria within 6 months of age. Patients 1 and 2 were siblings possessing a nonsense variant (c.9232C>T, p.(Arg3078*)) and a splice site variant (c.1282+1G>A) that led to exon 9 skipping and a frameshift. Patient 3 had a remarkable irregular contour of the glomerular basement membrane. She was subsequently found to have a nonsense variant (c.8185C>T, p.(Arg2720*)) and the same splice site variant in patients 1 and 2. By in vitro heterotrimer formation assays, both truncating variants produced smaller laminin α5 proteins that nevertheless formed trimers with laminin β1 and γ1 chains. Patient 4 showed SRNS at the age of eight and carried compound heterozygous missense variants (c.1493C>T, p.(Ala498Val) and c.8399G>A, p.(Arg2800His)). Conclusions: Our patients showed clear evidence of biallelic LAMA5 truncating variants causing infantile nephrotic syndrome. We also discerned the clinical and pathological characteristics observed in LAMA5-related nephropathy. LAMA5 variant screening should be performed in congenital/infantile nephrotic syndrome patients.

A Common Founder Effect of the Splice Site Variant c.-23+1G>A in GJB2 Gene Causing Autosomal Recessive Deafness 1A (DFNB1A) in Eurasia

The mutations in the GJB2 gene are known to be a major cause of autosomal recessive deafness 1A (OMIM 220290). The most common pathogenic variants of the GJB2 gene have high ethno-geographic specificity in their distribution that being attributed to a founder effect related with Neolithic migration routes of Homo sapiens. Curiously, the c.-23+1G>A splice site variant is frequently found among deaf patients of both Caucasian and Asian origin. It is currently unknown whether this mutation did spread across Eurasia as a result of the founder effect or it could have multiple local centers of origin. To determine the origin of the c.-23+1G>A we reconstructed 𝑓2-haplotypes by genotyping SNPs on the Illumina OmniExpress 730K platform in 23 deaf individuals homozygous for this variant from different populations of Eurasia (Yakuts, Tuvinians, Evenk, Kumyk, Armenian, Russians and Slovak). The analysis revealed that the homozygosity regions in different individuals overlapped in one short region with the length of ~5.2 kb. These data support the hypothesis of the common founder effect in distribution of the c.-23+1G>A variant of GJB2 gene. Based on the published data on the c.-23+1G>A prevalence among 16,177 deaf people and calculation of TMRCA of the 𝑓2-haplotypes carrying this variant we reconstructed the potential migration routes of the c.-23+1G>A carriers around the world. This analysis indicates that the c.-23+1G>A variant may have originated approximately 6,000 years ago in the territory of the Caucasus or Middle East, followed by spread throughout Europe, South and Central Asia and other regions of the world.

P–044 A non-classical splice site variant in ANOS1 gene leading to Kallmann syndrome

Study question Genetic risk of the non-classical splice site variant in ANOS1 gene Summary answer A non-classical ANOS1 splice site variant, c.1062 + 4T>C, causes Kallmann syndrome. What is known already Genetic abnormalities play a key role in the development of Kallmann syndrome. Although an overwhelming majority of missense and nonsense mutations occur in the exons of a gene, intron mutations can also be pathogenic. Study design, size, duration The research object is a family. Eight patients of the family were recruited in this study, three of them were diagnosed with Kallmann syndrome. Participants/materials, setting, methods Genomic DNA was extracted from peripheral blood and whole-exome sequencing (WES) was performed to identify the genetic abnormalities. PCR was performed to verify the WES results. The functional splicing reporter mini gene assay was performed to assess the impact of sequence variants on splicing. Main results and the role of chance The proband and other two patients exhibited the typical clinical features of KS. A non-classical splice site variant, c.1062 + 4T>C in ANOS1 gene was identified, whereas the other unaffected family members did not have this mutation. This mutation caused the disappearance of the splicing site of intron 7 and the splicing position became the 156th base of exon 7, which caused a frame-shift mutation, leading to a premature termination of translation. Limitations, reasons for caution Since the ANOS1 gene is almost not expressed in the blood, in order to uncover the effect of this splice site variant of ANOS1, we carried out a functional splicing reporter mini gene assay in the mini gene vector pEGFP-N1. Wider implications of the findings: This study shows that mutations in non-classical splicing regions are also pathogenic. Therefore, it is recommended that the detection and analysis of this gene should pay attention to the non-classical splice site variant. Trial registration number Not applicable