Two rare autosomal recessive neurological disorders identified by combined genetic approaches in a single consanguineous family with multiple offspring.

INTRODUCTION: Neurodevelopmental disorders (NDDs) refer to a broad range of diseases including developmental delay, intellectual disability, epilepsy, autism spectrum disorders, and attention-deficit/hyperactivity disorder caused by dysfunctions in tightly controlled brain development. The genetic backgrounds of NDDs are quite heterogeneous; to date, recessive or dominant variations in numerous genes have been implicated. Herein, we present a large consanguineous family from Turkiye, who has been suffering from NDDs with two distinct clinical presentations. METHODS AND RESULTS: Combined in-depth genetic approaches led us to identify a homozygous frameshift variant in NALCN related to NDD and expansion of dodecamer repeat in CSTB related to Unverricht-Lundborg disease (ULD). Additionally, we sought to functionally analyze the NALCN variant in terms of mRNA expression level and current alteration. We have both detected a decrease in the level of premature stop codon-bearing mRNA possibly through nonsense-mediated mRNA decay mechanism and also an increased current in patch-clamp recordings for the expressed truncated protein. CONCLUSION: In conclusion, increased consanguinity may lead to the revealing of distinct rare neurogenetic diseases in a single family. Exome sequencing is generally considered the first-tier diagnostic test in individuals with NDD. Yet we underline the fact that customized approaches other than exome sequencing may be used as in the case of ULD to aid diagnosis and better genetic counseling.

Targeted resequencing reveals high-level mosaicism for a novel frameshift variant in WDR45 associated with beta-propeller protein-associated neurodegeneration.

OBJECTIVES: Beta-propeller protein-associated neurodegeneration (BPAN) is a rare X-linked dominant neurodegenerative disease, which is characterized by iron accumulation in the basal ganglia. BPAN is associated with pathogenic variation in WDR45, which has been reported almost exclusively in females most probably due to male lethality in the hemizygous state. METHODS: Whole exome sequencing (WES) and targeted deep sequencing were performed for a male with a clinical diagnosis of BPAN at the age of 37. RESULTS: The novel frameshift variant in WDR45 detected by WES was further analyzed with targeted resequencing to detect a mosaic variant with a level of 85.5% in the blood sample of the proband. DISCUSSION: Although the main role of WDR45 remains elusive, recent studies show that WDR45 may contribute to neurodegeneration through defects in autophagy, iron storage and ferritin metabolism, mitochondria organization, and endoplasmic reticulum homeostasis. The extend of spatiotemporal haploinsufficiency of WDR45 frameshifting variants caused by mosaicism in males may lead to variable clinical severity, which may be hard to elaborate clinically. Promising genetic analysis strategies using targeted deep sequencing may help determine the clinical outcome of somatic mosaicism in neurological disorders including BPAN. Additionally, we suggest that deep sequencing should be conducted in cerebrospinal fluid samples to provide more reliable results in terms of reflecting the mosaicism level in the brain for future studies.

Epilepsy or neurodevelopmental disorders are associated with homozygous and pathogenic ELP2 variation in three siblings.

Developmental and Epileptic Encephalopathies (DEEs) are a group of early-onset syndromic disorders characterized by varying degree of intellectual disability, autism spectrum, seizures, and developmental delay. Herein, we have clinically and genetically dissected three siblings from Turkey with DEE born to first cousin unaffected parents. We identified a homozygous pathogenic variant in ELP2 (ENST00000358232.11:c.1385G>A; p.(Arg462Gln)). Our results, together with in depth literature review, underlie the importance of codon encoding the arginine at position 462 as a hotspot for ELP2 related neurological phenotypes.

Two cases with mitochondrial membrane protein-associated neurodegeneration: genetic features and long-term clinical follow-up.

Mitochondrial membrane protein-associated neurodegeneration (MPAN) is a rare neurological disease with childhood or adult onset. It is a subtype of clinically and genetically heterogeneous group of disorders, collectively known as neurodegeneration with brain iron accumulation . MPAN is generally associated with biallelic pathogenic variants in C19orf12. Herein, we describe genetic and clinical findings of two MPAN cases from Turkey. In the first case, we have identified the relatively common pathogenic variant of C19orf12 in the homozygous state, which causes late-onset MPAN. The second case was homozygous for an essential splice-site variation.

The rare rs769301934 variant in NHLRC1 is a common cause of Lafora disease in Turkey.

Lafora disease (LD) is a severe form of progressive myoclonus epilepsy inherited in an autosomal recessive fashion. It is associated with biallelic pathogenic variations in EPM2A or NHLRC1, which encode laforin and malin, respectively. The disease usually starts with adolescent onset seizures followed by progressive dementia, refractory status epilepticus and eventually death within 10 years of onset. LD is generally accepted as having a homogenous clinical course with no considerable differences between EPM2A or NHLRC1 associated forms. Nevertheless, late-onset and slow progressing forms of the disease have also been reported. Herein, we have performed clinical and genetic analyses of 14 LD patients from 12 different families and identified 8 distinct biallelic variations in these patients. Five of these variations were novel and/or associated with the LD phenotype for the first time. Interestingly, almost half of the cases were homozygous for the rare rs769301934 (NM_198586.3(NHLRC1): c.436 G > A; p.(Asp146Asn)) allele in NHLRC1. A less severe phenotype with an onset at a later age may be the reason for the biased inflation of this variant, which is already present in the human gene pool and can hence arise in the homozygous form in populations with increased parental consanguinity.

Biallelic loss of EEF1D function links heat shock response pathway to autosomal recessive intellectual disability.

Intellectual disability (ID) is a genetically heterogeneous neurodevelopmental disorder characterised by significantly impaired intellectual and adaptive functioning. ID is commonly syndromic and associated with developmental, metabolic and/or neurological findings. Autosomal recessive ID (ARID) is a significant component of ID especially in the presence of parental consanguinity. Several ultra rare ARID associated variants in numerous genes specific almost to single families have been identified by unbiased next generation sequencing technologies. However, most of these new candidate ARID genes have not been replicated in new families due to the rarity of associated alleles in this highly heterogeneous condition. To determine the genetic component of ARID in a consanguineous family from Turkey, we have performed SNP-based linkage analysis in the family along with whole exome sequencing (WES) in an affected sibling. Eventually, we have identified a novel pathogenic variant in EEF1D, which has recently been recognised as a novel candidate gene for ARID in a single family. EEF1D encodes a ubiquitously expressed translational elongation factor functioning in the cytoplasm. Herein, we suggest that the loss of function variants exclusively targeting the long EEF1D isoform may explicate the ARID phenotype through the heat shock response pathway, rather than interfering with the canonical translational elongation.

Clinical and genetic spectrum of an orphan disease MPAN: a series with new variants and a novel phenotype.

INTRODUCTION: Pathogenic variations in C19orf12 are responsible for two allelic diseases: mitochondrial membrane protein-associated neurodegeneration (MPAN); and spastic paraplegia type 43 (SPG43). MPAN is an orphan disease, which presents with spasticity, dystonia, peripheral nerve involvement, and dementia. The pattern of iron accumulation on brain MRI may be a clue for the diagnosis of MPAN. SPG43, on the other hand, is characterised by progressive lower limb spasticity without brain iron accumulation. We here present clinical and genetic findings of MPAN patients with potentially pathogenic C19orf12 variants. MATERIALS AND METHODS: Patients from 13 different families having progressive motor symptoms with irritative pyramidal signs and brain iron accumulation were screened for C19orf12 gene variants. RESULTS: C19orf12 screening identified seven variants associated with MPAN in eight patients from seven families. We associated two pathogenic variants (c.24G > C; p.(Lys8Asn) and c.194G > A; p.(Gly65Glu)) with the MPAN phenotype for the first time. We also provided a genetic diagnosis for a patient with an atypical MPAN presentation. The variant c.32C > T; p.(Thr11Met), common to Turkish adult-onset MPAN patients, was also detected in two unrelated late-onset MPAN patients. CONCLUSIONS: Genetic analysis along with thorough clinical analysis supported by radiological findings will aid the differential diagnosis of MPAN within the neurodegeneration with brain iron accumulation spectrum as well as other disorders including hereditary spastic paraplegia. Dystonia and parkinsonism may not be the leading clinical findings in MPAN patients, as these are absent in the atypical case. Finally, we emphasise that the existence of frameshifting variants may bias the age of onset toward childhood.

SYNE1 related cerebellar ataxia presents with variable phenotypes in a consanguineous family from Turkey.

SYNE1 related autosomal recessive cerebellar ataxia type 1 (ARCA1) is a late-onset cerebellar ataxia with slow progression originally demonstrated in French-Canadian populations of Quebec, Canada. Nevertheless, recent studies on SYNE1 ataxia have conveyed the condition from a geographically limited pure cerebellar recessive ataxia to a complex multisystem phenotype that is relatively common on the global scale. To determine the underlying genetic cause of the ataxia phenotype in a consanguineous family from Turkey presenting with very slow progressive cerebellar symptoms including dysarthria, dysmetria, and gait ataxia, we performed SNP-based linkage analysis in the family along with whole exome sequencing (WES) in two affected siblings. We identified a homozygous variant in SYNE1 (NM_033071.3: c.13086delC; p.His4362GlnfsX2) in all four affected siblings. This variant presented herein has originally been associated with only pure ataxia in a single case. We thus present segregation and phenotypic manifestations of this variant in four affected family members and further extend the pure ataxia phenotype with upper motor neuron involvement and peripheral neuropathy. Our findings in turn established a precise molecular diagnosis in this family, demonstrating the use of WES combined with linkage analysis in families as a powerful tool for establishing a quick and precise genetic diagnosis of complex neurological phenotypes.

An Extraordinary EEG Phenomenon Misdiagnosed as Nonconvulsive Status Epilepticus: Frequent Subclinical Periodic Discharges Terminated by Sudden Auditory Stimuli.

Triggering or modulation of seizures and rhythmic EEG patterns by external stimuli are well-known with the most common clinical appearance of stimulus induced periodic discharges (SI- PDs) patterns which are elicited by physical or auditory stimulation. However, stimulus terminated periodic discharges (ST-PDs), in other words, the periodic discharges stopped by external stimuli is an extremely rare electroencephalographic (EEG) finding. We report a 20-year-old woman with a marked psychomotor developmental delay of unknown cause, with frequent EEG patterns of long-lasting (10-60 s) bilateral paroxysmal high-voltage slow waves with occasional spikes, misdiagnosed as non-convulsive status epilepticus. However, no apparent clinical change was noted by the technician, physician, and her mother during these subclinical ictal EEG recordings. Interestingly, however, these epileptic discharges were abruptly interrupted by sudden verbal stimuli on the EEG, repeatedly. Whole exome sequencing and genotyping were performed to investigate possible genetic etiology that revealed two sequence variants, a frameshift variant of CACNA1H NM_021098.3:c.1701del;p.Asp568ThrfsTer15 and a missense variant of GRIN2D NM_000836.4:c.1783A>T;p.Thr595Ser as well as a copy number variant part deletion of ATP6V1A gene arr [hg19]3q13.31(113,499,698_113,543,081)x1 as possible pathogenic candidates. The subclinical periodic discharges terminated by verbal stimuli, is a very rare manifestation and needs particular attention. External modulation of ictal-appearing EEG patterns is important to identify stimulus terminated EEG patterns.

Clinical and genetic analyses in syndromic intellectual disability with primary microcephaly reveal biallelic and de novo variants in patients with parental consanguinity.

BACKGROUND: Syndromic intellectual disability (ID) with accompanying primary microcephaly is a group of rare neurodevelopmental disorders exhibiting extreme genetic and clinical heterogeneity. This layered heterogeneity can partially be resolved by unbiased genetic approaches targeting the genome with next generation sequencing (NGS) technologies, including exome sequencing (ES). OBJECTIVE: This study was performed to dissect the clinical and genetic features in five distinct IDM cases. METHODS: Singleton or trio ES approach followed by in-depth variant analysis using alternative inheritance models was performed. RESULTS: We have identified biallelic loss of function variants in genes WDR62 and AP4M1 in three families, together with de novo missense variants in genes SOX11 and TRIO in two families. ES based haplotype analysis in two cases upon identification of an identical WDR62 variant in the homozygous state in two cases was suggestive of a small shared haplotype of 0.1 Mb. Additionally, we have shown a paternal origin for the de novo variant in TRIO via a polymorphic tag SNP, which enlightens the mutational mechanism for this variant. CONCLUSION: In populations with high parental consanguinity, an autosomal recessive inheritance pattern for data analysis is usually the most obvious choice. Therefore, heterozygous variants may be overlooked in standard NGS analyses in consanguineous families. Our findings underlie the importance of using multiple inheritance models in NGS data analysis.

Obstacles and expectations of rare disease patients and their families in Türkiye: ISTisNA project survey results.

Rare disease patients constitute a significant part of the healthcare system of all countries. However, the information on the experiences during disease processes and daily life of rare disease patients is still limited. So far, there is a small number of studies conducted in Türkiye, and they mainly cover specific issues like education or anxiety. Here we present a comprehensive survey analysis conducted among the patients and their families within the scope of the Istanbul Solution Platform for Undiagnosed and Rare Diseases-ISTisNA project. A total of 498 individuals responded to the survey, and 58% of the participants answered all questions. The majority of the patients were in the age range of 1-10 years (44.7%), and 91% of all the patients had a precise diagnosis. The diagnosis rate in the first 6 months was 69%, and almost 10% of the patients remained undiagnosed. The mothers were the primary caregivers (72%). Nearly 30% of the caregivers had to quit their jobs and 25% of the patients (0-18 years) had to leave school. Accessing physicians with relevant specialization and reaching treatments/medications/supplements were the two main obstacles the participants mentioned, with a frequency of 81% and 73%, respectively. Around 50% of participants noted that they commonly faced difficulties at work/school and in their social lives. The highest expectation or priority was the establishment of rare disease-specific diagnosis and treatment centers, accurate and detailed information on diseases in the Turkish language, and easy access to physicians, treatments, and supportive therapies. To the best of our knowledge, this is the most comprehensive survey conducted on the rare disease community in Türkiye. These results show that regardless of the country, the individuals affected by rare diseases and their families have similar problems and expectations. On the other hand, regional and country-specific issues are still in the line to be solved. These studies can provide a deeper insight into rare diseases and guide the activities of Türkiye's national rare disease action plan.

Identification of epilepsy related pathways using genome-wide DNA methylation measures: A trio-based approach.

Genetic generalized epilepsies (GGE) are genetically determined, as their name implies and they are clinically characterized by generalized seizures involving both sides of the brain in the absence of detectable brain lesions or other known causes. GGEs are yet complex and are influenced by many different genetic and environmental factors. Methylation specific epigenetic marks are one of the players of the complex epileptogenesis scenario leading to GGE. In this study, we have set out to perform genome-wide methylation profiling to analyze GGE trios each consisting of an affected parent-offspring couple along with an unaffected parent. We have developed a novel scoring scheme within trios to categorize each locus analyzed as hypo or hypermethylated. This stringent approach classified differentially methylated genes in each trio and helped us to produce trio specific and pooled gene lists with inherited and aberrant methylation levels. In order to analyze the methylation differences from a boarder perspective, we performed enrichment analysis with these lists using the PANOGA software. This collective effort has led us to detect pathways associated with the GGE phenotype, including the neurotrophin signaling pathway. We have demonstrated a trio based approach to genome-wide DNA methylation analysis that identified individual and possibly minor changes in methylation marks that could be involved in epileptogenesis leading to GGE.

A Novel and Mosaic WDR45 Nonsense Variant Causes Beta-Propeller Protein-Associated Neurodegeneration Identified Through Whole Exome Sequencing and X chromosome Heterozygosity Analysis.

Beta-propeller protein-associated neurodegeneration (BPAN) is an X-linked rare dominant disorder of autophagy. The role of WDR45 has been implicated in BPAN almost exclusively in females possibly due to male lethality. Characterization of distinctive clinical manifestations and potentially the complex genetic determinants in rare male patients remain crucial for deciphering BPAN and other X-linked dominant diseases. We performed whole exome sequencing (WES) followed by segregation analysis and identified a novel nonsense and mosaic variant in WDR45, namely NM_007075.3:c.873C>G; p.(Tyr291*) in an affected male at the age of 34. His biphasic medical history was compatible with BPAN, which was characterized by delayed psychomotor development, intellectual disability, and progression into dystonia parkinsonism in his twenties. The variant had an apparently mosaic pattern both in whole exome and Sanger sequencing findings. In order to figure out if mosaicism was restricted to this variant or related to a chromosomal level mosaicism, we used our in-house WES data from 129 unrelated individuals to calculate the threshold values of male and female X chromosome heterozygosity (XcHet) in WES data for our pipeline. A background level of heterozygous variants on X chromosome excluding the pseudoautosomal loci is an observed phenomenon in WES analysis and this level has been used as a quality measure. Herein, we suggest utilization of this measure for detection of digital anomalies of the X chromosome in males by potentially observing a higher XcHet value than the threshold value. This approach has revealed a variant level mosaicism in the affected male, which was further supported with cytogenetic analyses.

Investigation of SLC2A1 gene variants in genetic generalized epilepsy patients with eyelid myoclonia.

In addition to a complex inheritance pattern in genetic generalized epilepsy (GGE) syndromes, some studies have recently identified SLC2A1 variants which lead to glucose transporter type 1 (GLUT1) defects, in patients diagnosed with GGE. Here, we investigated the possible role of SLC2A1 variants in GGE patients with eyelid myoclonia (EM) which is a rare generalized seizure type associated with drug resistance and cognitive dysfunction. After polymerase chain reaction with designed primers, sequencing of all SLC2A1 exons was performed for 25 GGE-EM patients, as well as a control group of 15 GGE patients with absence seizures. Although various single nucleotide polymorphisms clustered in the ninth exon were detected, no variant was found in the two groups with GGE. Even though the patient number in this study is small, the data suggest that SLC2A1 variants do not play any causative role in GGE associated with EM.

A clinical variant in SCN1A inherited from a mosaic father cosegregates with a novel variant to cause Dravet syndrome in a consanguineous family.

A consanguineous family from Turkey having two children with intellectual disability exhibiting myoclonic, febrile and other generalized seizures was recruited to identify the genetic origin of these phenotypes. A combined approach of SNP genotyping and exome sequencing was employed both to screen genes associated with Dravet syndrome and to detect homozygous variants. Analysis of exome data was extended further to identify compound heterozygosity. Herein, we report identification of two paternally inherited genetic variants in SCN1A (rs121917918; p.R101Q and p.I1576T), one of which was previously implicated in Dravet syndrome. Interestingly, the previously reported clinical variant (rs121917918; p.R101Q) displayed mosaicism in the blood and saliva of the father. The study supported the genetic diagnosis of affected children as Dravet syndrome possibly due to the combined effect of one clinically associated (rs121917918; p.R101Q) and one novel (p.I1576T) variants in SCN1A gene. This finding is important given that heterozygous variants may be overlooked in standard exome scans of consanguineous families. Thus, we are presenting an interesting example, where the inheritance of the condition may be misinterpreted as recessive and identical by descent due to consanguinity and mosaicism in one of the parents.

A novel recessive GUCY2D mutation causing cone-rod dystrophy and not Leber's congenital amaurosis.

Cone-rod dystrophies are inherited retinal dystrophies that are characterized by progressive degeneration of cones and rods, causing an early decrease in central visual acuity and colour vision defects, followed by loss of peripheral vision in adolescence or early adult life. Both genetic and clinical heterogeneity are well known. In a family with autosomal recessive cone-rod dystrophy, genetic analyses comprising genome scan with microsatellite markers, fine mapping and candidate gene approach resulted in the identification of a homozygous missense GUCY2D mutation. This is the first GUCY2D mutation associated with autosomal recessive cone-rod dystrophy rather than Leber's congenital amaurosis (LCA), a severe disease leading to childhood blindness. This study hence establishes GUCY2D, which is a common cause for both recessive LCA and dominant cone-rod dystrophy, as a good candidate for autosomal recessive cone-rod dystrophy.