Margit Nõukas

De novo SCN8A mutation identified by whole-exome sequencing in a boy with neonatal epileptic encephalopathy, multiple congenital anomalies, and movement disorders.

Epileptic encephalopathies represent a clinically and genetically heterogeneous group of disorders, majority of which are of unknown etiology. We used whole-exome sequencing of a parent-offspring trio to identify the cause of early infantile epileptic encephalopathy in a boy with neonatal seizures, movement disorders, and multiple congenital anomalies who died at the age of 17 months because of respiratory illness and identified a de novo heterozygous missense mutation (c.3979A>G; p.Ile1327Val) in SCN8A (voltage-gated sodium-channel type VIII alpha subunit) gene. The variant was confirmed in the proband with Sanger sequencing. Because the clinical phenotype associated with SCN8A mutations has previously been identified only in a few patients with or without epileptic seizures, these data together with our results suggest that mutations in SCN8A can lead to early infantile epileptic encephalopathy with a broad phenotypic spectrum. Additional investigations will be worthwhile to determine the prevalence and contribution of SCN8A mutations to epileptic encephalopathies.

Coffin–Siris Syndrome with obesity, macrocephaly, hepatomegaly and hyperinsulinism caused by a mutation in the ARID1B gene

Coffin–Siris Syndrome (CSS, MIM 135900) is a rare genetic disorder, and mutations in ARID1B were recently shown to cause CSS. In this study, we report a novel ARID1B mutation identified by whole-exome sequencing in a patient with clinical features of CSS. We identified a novel heterozygous frameshift mutation c.1584delG in exon 2 of ARID1B (NM_020732.3) predicting a premature stop codon p.(Leu528Phefs*65). Sanger sequencing confirmed the c.1584delG mutation as a de novo in the proband and that it was not present either in her parents, half-sister or half-brother. Clinically, the patient presented with extreme obesity, macrocephaly, hepatomegaly, hyperinsulinism and polycystic ovarian syndrome (PCOS), which have previously not been described in CSS patients. We suggest that obesity, macrocephaly, hepatomegaly and/or PCOS may be added to the list of clinical features of ARID1B mutations, but further clinical reports are required to make a definite conclusion.

Syndromic intellectual disability: A new phenotype caused by an aromatic amino acid decarboxylase gene (DDC) variant

The causative variant in a consanguineous family in which the three patients (two siblings and a cousin) presented with intellectual disability, Marfanoid habitus, craniofacial dysmorphisms, chronic diarrhea and progressive kyphoscoliosis, has been identified through whole exome sequencing (WES) analysis. WES study identified a homozygous DDC variant in the patients, c.1123C>T, resulting in p.Arg375Cys missense substitution. Mutations in DDC cause a recessive metabolic disorder (aromatic amino acid decarboxylase, AADC, deficiency, OMIM #608643) characterized by hypotonia, oculogyric crises, excessive sweating, temperature instability, dystonia, severe neurologic dysfunction in infancy, and specific abnormalities of neurotransmitters and their metabolites in the cerebrospinal fluid (CSF). In our family, analysis of neurotransmitters and their metabolites in patients CSF shows a pattern compatible with AADC deficiency, although the clinical signs are different from the classic form. Our work expands the phenotypic spectrum associated with DDC variants, which therefore can cause an additional novel syndrome without typical movement abnormalities.

Copy number variation analysis detects novel candidate genes involved in follicular growth and oocyte maturation in a cohort of premature ovarian failure cases.

STUDY QUESTION Can spontaneous premature ovarian failure (POF) patients derived from population-based biobanks reveal the association between copy number variations (CNVs) and POF? SUMMARY ANSWER CNVs can hamper the functional capacity of ovaries by disrupting key genes and pathways essential for proper ovarian function. WHAT IS KNOWN ALREADY POF is defined as the cessation of ovarian function before the age of 40 years. POF is a major reason for female infertility, although its cause remains largely unknown. STUDY DESIGN, SIZE, DURATION The current retrospective CNV study included 301 spontaneous POF patients and 3188 control individuals registered between 2003 and 2014 at Estonian Genome Center at the University of Tartu (EGCUT) biobank. PARTICIPANTS/MATERIALS, SETTING, METHODS DNA samples from 301 spontaneous POF patients were genotyped by Illumina HumanCoreExome (258 samples) and HumanOmniExpress (43 samples) BeadChip arrays. Genotype and phenotype information was drawn from the EGCUT for the 3188 control population samples, previously genotyped with HumanCNV370 and HumanOmniExpress BeadChip arrays. All identified CNVs were subjected to functional enrichment studies for highlighting the POF pathogenesis. Real-time quantitative PCR was used to validate a subset of CNVs. Whole-exome sequencing was performed on six patients carrying hemizygous deletions that encompass genes essential for meiosis or folliculogenesis. MAIN RESULTS AND THE ROLE OF CHANCE Eleven novel microdeletions and microduplications that encompass genes relevant to POF were identified. For example, FMN2 (1q43) and SGOL2 (2q33.1) are essential for meiotic progression, while TBP (6q27), SCARB1 (12q24.31), BNC1 (15q25) and ARFGAP3 (22q13.2) are involved in follicular growth and oocyte maturation. The importance of recently discovered hemizygous microdeletions of meiotic genes SYCE1 (10q26.3) and CPEB1 (15q25.2) in POF patients was also corroborated. LIMITATIONS, REASONS FOR CAUTION This is a descriptive analysis and no functional studies were performed. Anamnestic data obtained from population-based biobank lacked clinical, biological (hormone levels) or ultrasonographical data, and spontaneous POF was predicted retrospectively by excluding known extraovarian causes for premature menopause. WIDER IMPLICATIONS OF THE FINDINGS The present study, with high number of spontaneous POF cases, provides novel data on associations between the genomic aberrations and premature menopause of ovarian cause and demonstrates that population-based biobanks are powerful source of biological samples and clinical data to reveal novel genetic lesions associated with human reproductive health and disease, including POF. STUDY FUNDING/COMPETING INTEREST This study was supported by the Estonian Ministry of Education and Research (IUT20-43, IUT20-60, IUT34-16, SF0180027s10 and 9205), Enterprise Estonia (EU30020 and EU48695), Eurekas EUROSTARS programme (NOTED, EU41564), grants from European Unions FP7 Marie Curie Industry-Academia Partnerships and Pathways (IAPP, SARM, |EU324509) and Horizon 2020 innovation programme (WIDENLIFE, 692065), Academy of Finland and the Sigrid Juselius Foundation.

A parallel SNP array study of genomic aberrations associated with mental retardation in patients and general population in Estonia

The increasing use of whole-genome array screening has revealed the important role of DNA copy-number variations in the pathogenesis of neurodevelopmental disorders and several recurrent genomic disorders have been defined during recent years. However, some variants considered to be pathogenic have also been observed in phenotypically normal individuals. This underlines the importance of further characterization of genomic variants with potentially variable expressivity in both patient and general population cohorts to clarify their phenotypic consequence. In this study whole-genome SNP arrays were used to investigate genomic rearrangements in 77 Estonian families with idiopathic mental retardation. In addition to this family-based approach, phenotype and genotype data from a cohort of 1000 individuals in the general population were used for accurate interpretation of aberrations found in mental retardation patients. Relevant structural aberrations were detected in 18 of the families analyzed (23%). Fifteen of those were in genomic regions where clinical significance has previously been established. In 3 families, 4 novel aberrations associated with intellectual disability were detected in chromosome regions 2p25.1-p24.3, 3p12.1-p11.2, 7p21.2-p21.1 and Xq28. Carriers of imbalances in 15q13.3, 16p11.2 and Xp22.31 were identified among reference individuals, affirming the variable phenotypic consequence of rare variants in some genomic regions considered as pathogenic.

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.

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.

Whole-exome sequencing identifies de novo mutation in the COL1A1 gene to underlie the severe osteogenesis imperfecta

BackgroundOsteogenesis imperfecta (OI) comprises a clinically and genetically heterogeneous group of connective tissue disorders, characterized by low bone mass, increased bone fragility, and blue-gray eye sclera. OI often results from missense mutations in one of the conserved glycine residues present in the Gly-X-Y sequence repeats of the triple helical region of the collagen type I α chain, which is encoded by the COL1A1 gene. The aim of the present study is to describe the phenotype of OI II patient and a novel mutation, causing current phenotype.ResultsWe report an undescribed de novo COL1A1 mutation in a patient affected by severe OI. After performing the whole-exome sequencing in a case parent–child trio, we identified a novel heterozygous c.2317G > T missense mutation in the COL1A1 gene, which leads to p.Gly773Cys transversion in the triple helical domain of the collagen type I α chain. The presence of the missense mutation was confirmed with the Sanger sequencing.ConclusionsHereby, we report a novel mutation in the COL1A1 gene causing severe, life threatening OI and indicate the role of de novo mutation in the pathogenesis of rare familial diseases. Our study underlines the importance of exome sequencing in disease gene discovery for families where conventional genetic testing does not give conclusive evidence.

Reporting incidental findings of genomic disorder-associated copy number variants to unselected biobank participants

BACKGROUND Procedural guidelines for disclosure of incidental genomic information are lacking. METHODS We introduce a method and evaluated the impact of returning results to population biobank participants with 16p11.2 copy number variants, which are commonly associated with neurodevelopmental disorders and BMI imbalance. Of the 7877 participants, 11 carriers were detected. Eight participants were informed of their carrier status and surveyed 11-17 months later. RESULTS All participants demonstrated preference for disclosure. Although two participants experienced worry, all five survey respondents rated receiving this information favorably. One participant reported modifications in treatment and three felt that their treatment/condition had since improved. CONCLUSION This approach can be adapted and applied for the return of incidental findings to biobank participants.

C14orf132 gene is possibly related to extremely low birth weight

BackgroundDespite extensive research the genetic component of extremely low birth weight (ELBW) in newborns has remained obscure.ResultsThe aim of the case study was to identify candidate gene(s) causing ELBW in newborns and hypotrophy in infants. A family of four was studied: mother, father and two ELBW-phenotype children. Studies were made of the medical conditions of the second child at birth and post-partum - peculiar phenotype, micro-anomalies, recurrent infections, suspicion of autoimmune hepatitis, multifactorial encephalopathy and suspected metabolic and chromosomal abnormalities. Whole genome single nucleotide polymorphism (SNP) genotyping array was used to investigate the genomic rearrangements in both affected children using peripheral blood DNA samples. Whole blood transcriptome was assessed by using RNA sequencing (RNA-seq) in all four family members. RNA-seq identified a single gene – C14orf132 (chromosome 14 open reading frame 132) differentially expressed, with the level of the transcript significantly lower in the blood samples of the children. Copy number variant (CNV) analysis did not reveal any pathogenic CNVs in the region of C14orf132 gene of both affected children.ConclusionWe demonstrated the importance of combining whole genome CNV and transcriptome analysis in identification of the candidate gene(s) in case studies. We propose the C14orf132 gene expression to be associated with the ELBW-phenotype. C14orf132 gene is a novel long non-coding RNA (lincRNA) with unknown function, which might be associated with the pre- and early postnatal developmental delay through the altered gene expression.