Sander Pajusalu

Biallelic CACNA1A mutations cause early onset epileptic encephalopathy with progressive cerebral, cerebellar, and optic nerve atrophy.

The CACNA1A gene encodes the transmembrane pore‐forming alpha‐1A subunit of the Cav2.1 P/Q‐type voltage‐gated calcium channel. Several heterozygous mutations within this gene, including nonsense mutations, missense mutations, and expansion of cytosine‐adenine‐guanine repeats, are known to cause three allelic autosomal dominant conditions—episodic ataxia type 2, familial hemiplegic migraine type 1, and spinocerebellar ataxia type 6. An association with epilepsy and CACNA1A mutations has also been described. However, the link with epileptic encephalopathies has emerged only recently. Here we describe two patients, sister and brother, with compound heterozygous mutations in CACNA1A. Exome sequencing detected biallelic mutations in CACNA1A: A missense mutation c.4315T>A (p.Trp1439Arg) in exon 27, and a seven base pair deletion c.472_478delGCCTTCC (p.Ala158Thrfs*6) in exon 3. Both patients were normal at birth, but developed daily recurrent seizures in early infancy with concomitant extreme muscular hypotonia, hypokinesia, and global developmental delay. The brain MRI images showed progressive cerebral, cerebellar, and optic nerve atrophy. At the age of 5, both patients were blind and bedridden with a profound developmental delay. The elder sister died at that age. Their parents and two siblings were heterozygotes for one of those pathogenic mutations and expressed a milder phenotype. Both of them have intellectual disability and in addition the mother has adult onset cerebellar ataxia with a slowly progressive cerebellar atrophy. Compound heterozygous mutations in the CACNA1A gene presumably cause early onset epileptic encephalopathy, and progressive cerebral, cerebellar and optic nerve atrophy with reduced lifespan.

Diffuse hypomyelination is not obligate for POLR3-related disorders.

Objective: To report atypical MRI patterns associated with POLR3A and POLR3B mutations. Methods: This was a multicenter retrospective study to collect neuroradiologic, clinical, and molecular data of patients with mutations in POLR3A and POLR3B without the classic MRI phenotype, i.e., diffuse hypomyelination associated with relative T2 hypointensity of the ventrolateral thalamus, globus pallidus, optic radiation, corticospinal tract at the level of the internal capsule, and dentate nucleus, cerebellar atrophy, and thinning of the corpus callosum. Results: Eight patients were identified: 6 carried mutations in POLR3A and 2 in POLR3B. We identified 2 novel MRI patterns: 4 participants presented a selective involvement of the corticospinal tracts, specifically at the level of the posterior limbs of the internal capsules; 4 patients presented moderate to severe cerebellar atrophy. Incomplete hypomyelination was observed in 5 participants. Conclusion: Diffuse hypomyelination is not an obligatory feature of POLR3-related disorders. Two distinct patterns, selective involvement of the corticospinal tracts and cerebellar atrophy, are added to the MRI presentation of POLR3-related disorders.

Novel homozygous mutation in KPTN gene causing a familial intellectual disability‐macrocephaly syndrome

Recently, a novel autosomal recessive developmental delay‐macrocephaly syndrome was described caused by homozygous or compound heterozygous mutations in the KPTN gene. All reported patients belonged to one large Amish kindred. We report on the second case of KPTN‐related syndrome in two Estonian adult sibs. The brother and sister both have macrocephaly and moderate intellectual disability, and their verbal abilities are more affected than motor development. No notable minor anomalies are present. Behavioral problems and a few episodes of seizures were reported in the brother. Whole exome sequencing carried out from the brothers DNA sample identified homozygous one‐nucleotide frameshift duplication c.665dupA (p.Q222fs) in the KPTN gene. Homozygosity of both affected sibs and heterozygosity of parents were confirmed by Sanger sequencing. Thus, we confirm the pathogenicity of KPTN mutations and further delineate the novel developmental delay‐macrocephaly syndrome. We also support the hypothesis that KPTN‐related syndrome is not restricted to the Amish population.

Large gene panel sequencing in clinical diagnostics-results from 501 consecutive cases: PAJUSALU et al.

In addition to whole exomes, large gene panels of clinically associated genes are used as high‐throughput sequencing tests in many clinical centers, but their clinical utility has been much less investigated.

The Diagnostic Utility of Single Long Contiguous Stretches of Homozygosity in Patients without Parental Consanguinity

We present data from our clinical departments experience with chromosomal microarray analysis (CMA) regarding the diagnostic utility of 1 or 2 long contiguous stretches of homozygosity (LCSHs) in an outbred population. The study group consisted of 2,110 consecutive patients from 2011 to 2014 for whom CMA was performed. The minimum cut-off size for defining a homozygous stretch was 5 Mb. To focus on cases with no parental consanguinity, we further studied only patients in whom the total length of homozygous stretches did not exceed 28 Mb or 1% of the autosomal genome length. We identified 6 chromosomal regions where homozygous stretches appeared in at least 3 patients and excluded these from further analysis. In 2 out of 120 patients with an isolated finding of 1 or 2 non-recurrent LCSHs, a plausible candidate gene associated with their phenotype was identified within the homozygous stretch. In both of these cases, a pathogenic mutation was detected, leading to diagnoses of pyruvate kinase deficiency and Marinesco-Sjögren syndrome. To clarify whether previously found homozygous stretches could be important for the interpretation of genome-wide sequencing data, we report 7 cases in which homozygous stretches not encompassing a clinically associated gene were first found on CMA, followed by the diagnostic whole-exome sequencing. The diagnostic utility of single LCSHs, unlikely to be caused by uniparental disomy, is discussed in detail.

Three families with mild PMM2-CDG and normal cognitive development

Congenital disorders of glycosylation (CDG) are caused by defective glycosylation of proteins and lipids. PMM2‐CDG is the most common subtype among the CDG. The severity of PMM2‐CDG is variable. Patients often have a recognizable phenotype with neurological and multisystem symptoms that might cause early death. We report six patients from three families who are diagnosed with a clinically mild PMM2‐CDG and have normal cognitive development. All these patients had delayed gross motor skills with mild‐to‐moderate neurological findings. Cerebellar hypoplasia was detected in all siblings for whom brain MRI was performed. In 5/6 children the Wechsler Intelligence Scale for Children (WISC) showed normal cognitive development with full scale IQ scores ranging from borderline to average. Four patients were diagnosed with PMM2‐CDG at the age of 8 years or later as their neurological symptoms were quite mild and they had been able to participate in regular school programs. We report patients with p.Val231Met/p.Arg239Trp and p.Ile120Thr/p.Gly228Cys genotypes which may cause milder variants of PMM2‐CDG.

De novo exonic mutation in MYH7 gene leading to exon skipping in a patient with early onset muscular weakness and fiber-type disproportion

Here we report on a case of MYH7-related myopathy in a boy with early onset of muscular weakness and delayed motor development in infancy. His most affected muscles were neck extensors showing a dropped head sign, proximal muscles of lower limbs with positive Gowers sign, and trunk muscles. Brain and spinal cord MRI scans, echocardiography, and laboratory analyses including creatine kinase and lactate did not reveal any abnormalities. Muscle histopathology showed fiber-type disproportion. Whole exome sequencing of the parents-offspring trio revealed a novel de novo c.5655G>A p.(Ala1885=) synonymous substitution of the last nucleotide in exon 38 of the MYH7 gene. Further RNA investigations proved the skipping of exon 38 (p.1854_1885del). This is a first report of an exon-skipping mutation in the MYH7 gene causing myopathy. This report broadens both the phenotypic and genotypic spectra of MYH7-related myopathies.

Hippocampus and Hypothalamus RNA-sequencing of WFS1-deficient Mice

Wolfram syndrome is caused by mutations in the WFS1 gene. WFS1 protein dysfunction results in a range of neuroendocrine syndromes and is mostly characterized by juvenile-onset diabetes mellitus and optic atrophy. WFS1 has been shown to participate in membrane trafficking, protein processing and Ca2+ homeostasis in the endoplasmic reticulum. Aim of the present study was to find the transcriptomic changes influenced by WFS1 in the hypothalamus and hippocampus using RNA-sequencing. The WFS1-deficient mice were used as a model system to analyze the changes in transcriptional networks. The number of differentially expressed genes between hypothalami of WFS1-deficient (Wfs1KO) and wild-type (WT) mice was 43 and between hippocampi 311 with False Discovery Rate (FDR) <0.05. Avpr1a and Avpr1b were significantly upregulated in the hypothalamus and hippocampus of Wfs1KO mice respectively. Trpm8 was the most upregulated gene in the hippocampus of Wfs1KO mice. The functional analysis revealed significant enrichment of networks and pathways associated with protein synthesis, cell-to-cell signaling and interaction, molecular transport, metabolic disease and nervous system development and function. In conclusion, the transcriptomic profiles of WFS1-deficient hypothalamus and hippocampus do indicate the activation of degenerative molecular pathways causing the clinical occurrences typical to Wolfram syndrome.

Effectiveness of whole exome sequencing in unsolved patients with a clinical suspicion of a mitochondrial disorder in Estonia

Objective Reaching a genetic diagnosis of mitochondrial disorders (MDs) is challenging due to their broad phenotypic and genotypic heterogeneity. However, there is growing evidence that the use of whole exome sequencing (WES) for diagnosing patients with a clinical suspicion of an MD is effective (39–60%). We aimed to study the effectiveness of WES in clinical practice in Estonia, in patients with an unsolved, but suspected MD. We also show our first results of mtDNA analysis obtained from standard WES reads. Methods Retrospective cases were selected from a database of 181 patients whose fibroblast cell cultures had been stored from 2003 to 2013. Prospective cases were selected during the period of 2014–2016 from patients referred to a clinical geneticist in whom an MD was suspected. We scored each patient according to the mitochondrial disease criteria (MDC) (Morava et al., 2006) after re-evaluation of their clinical data, and then performed WES analysis. Results A total of 28 patients were selected to the study group. A disease-causing variant was found in 16 patients (57%) using WES. An MD was diagnosed in four patients (14%), with variants in the SLC25A4, POLG, SPATA5, and NDUFB11 genes. Other variants found were associated with a neuromuscular disease (SMN1, MYH2, and LMNA genes), neurodegenerative disorder (TSPOAP1, CACNA1A, ALS2, and SCN2A genes), multisystemic disease (EPG5, NKX1–2, ATRX, and ABCC6 genes), and one in an isolated cardiomyopathy causing gene (MYBPC3). The mtDNA point mutation was found in the MT-ATP6 gene of one patient upon mtDNA analysis. Conclusions The diagnostic yield of WES in our cohort was 57%, proving to be a very good effectiveness. However, MDs were found in only 14% of the patients. We suggest WES analysis as a first-tier method in clinical genetic practice for children with any multisystem, neurological, and/or neuromuscular problem, as nuclear DNA variants are more common in children with MDs; a large number of patients harbor disease-causing variants in genes other than the mitochondria-related ones, and the clinical presentation might not always point towards an MD. We have also successfully conducted analysis of mtDNA from standard WES reads, providing further evidence that this method could be routinely used in the future.

A New Case of a Rare Combination of Temple Syndrome and Mosaic Trisomy 14 and a Literature Review

Temple syndrome (TS14) is a relatively recently discovered imprinting disorder caused by abnormal expression of genes at the locus 14q32. The underlying cause of this syndrome is maternal uniparental disomy of chromosome 14 (UPD(14)mat). Trisomy of chromosome 14 is one of the autosomal trisomies; in humans, it is only compatible with live birth in mosaic form. Although UPD(14)mat and mosaic trisomy 14 can arise from the same cellular mechanism, a combination of both has been currently reported only in 8 live-born cases. Hereby, we describe a patient in whom only UPD(14)mat-associated TS14 was primarily diagnosed. Due to the patients atypical features (for TS14), additional analyses were performed and low-percent mosaic trisomy 14 was detected. It can be expected that the described combination of 2 etiologically related conditions is actually more prevalent. Additional chromosomal and molecular investigations are indicated for every patient with UPD(14)mat-associated TS14 with atypical clinical presentation.