Revealing parental mosaicism: the hidden answer to the recurrence of apparent de novo variants.

Mosaicism refers to the presence of two or more populations of genetically distinct cells within an individual, all of which originate from a single zygote. Previous literature estimated the percentage of parental mosaicism ranged from 0.33 to 25.9%. In this study, parents whose children had previously been diagnosed with developmental disorders with an apparent de novo variant were recruited. Peripheral blood, buccal and semen samples were collected from these parents if available for the detection of potential parental mosaicism using droplet digital PCR, complemented with the method of blocker displacement amplification. Among the 20 families being analyzed, we report four families with parental mosaicism (4/20, 20%). Two families have maternal gonosomal mosaicism (EYA1 and EBF3) and one family has paternal gonadal mosaicism (CHD7) with a pathogenic/ likely pathogenic variant. One family has a paternal gonosomal mosaicism with a variant of uncertain significance (FLNC) with high clinical relevance. The detectable variant allele frequency in our cohort ranged from 8.7-35.9%, limit of detection 0.08-0.16% based on our in-house EBF3 assay. Detecting parental mosaicism not only informs family with a more accurate recurrence risk, but also facilitates medical teams to create appropriate plans for pregnancy and delivery, offering the most suitable care.

Evaluating High-Confidence Genes in Conotruncal Cardiac Defects by Gene Burden Analyses.

Background In nonsyndromic conotruncal cardiac defects, the use of next-generation sequencing for clinical diagnosis is increasingly adopted, but gene-disease associations in research are only partially translated to diagnostic panels, suggesting a need for evidence-based consensus. Methods and Results In an exome data set of 245 patients with conotruncal cardiac defects, we performed burden analysis on a high-confidence congenital heart disease gene list (n=132) with rare (<0.01%) and ultrarare (absent in the Genome Aggregation Database) protein-altering variants. Overall, we confirmed an excess of rare variants compared with ethnicity-matched controls and identified 2 known genes (GATA6, NOTCH1) and 4 candidate genes supported by the literature (ANKRD11, DOCK6, NPHP4, and STRA6). Ultrarare variant analysis was performed in combination with 3 other published studies (n=1451) and identified 3 genes (FLT4, NOTCH1, TBX1) to be significant, whereas a subgroup analysis involving 391 Chinese subjects identified only GATA6 as significant. Conclusions We suggest that these significant genes in our rare and ultrarare burden analyses warrant prioritization for clinical testing implied for rare inherited and de novo variants. Additionally, associations on ClinVar for these genes were predominantly variants of uncertain significance. Therefore, a more stringent assessment of gene-disease associations in a larger and ethnically diverse cohort is required to be prudent for future curation of conotruncal cardiac defect genes.

Diagnostic potential of the amniotic fluid cells transcriptome in deciphering mendelian disease: a proof-of-concept.

RNA sequencing (RNA-seq) is emerging in genetic diagnoses as it provides functional support for the interpretation of variants of uncertain significance. However, the use of amniotic fluid (AF) cells for RNA-seq has not yet been explored. Here, we examined the expression of clinically relevant genes in AF cells (n = 48) compared with whole blood and fibroblasts. The number of well-expressed genes in AF cells was comparable to that in fibroblasts and much higher than that in blood across different disease categories. We found AF cells RNA-seq feasible and beneficial in prenatal diagnosis (n = 4) as transcriptomic data elucidated the molecular consequence leading to the pathogenicity upgrade of variants in CHD7 and COL1A2 and revising the in silico prediction of a variant in MYRF. AF cells RNA-seq could become a reasonable choice for postnatal patients with advantages over fibroblasts and blood as it prevents invasive procedures.

Delineation of molecular findings by whole-exome sequencing for suspected cases of paediatric-onset mitochondrial diseases in the Southern Chinese population.

BACKGROUND: Mitochondrial diseases (MDs) are a group of clinically and genetically heterogeneous disorders characterized by defects in oxidative phosphorylation. Since clinical phenotypes of MDs may be non-specific, genetic diagnosis is crucial for guiding disease management. In the current study, whole-exome sequencing (WES) was performed for our paediatric-onset MD cohort of a Southern Chinese origin, with the aim of identifying key disease-causing variants in the Chinese patients with MDs. METHODS: We recruited Chinese patients who had paediatric-onset MDs and a minimum mitochondrial disease criteria (MDC) score of 3. Patients with positive target gene or mitochondrial DNA sequencing results were excluded. WES was performed, variants with population frequency ≤ 1% were analysed for pathogenicity on the basis of the American College of Medical Genetics and Genomics guidelines. RESULTS: Sixty-six patients with pre-biopsy MDC scores of 3-8 were recruited. The overall diagnostic yield was 35% (23/66). Eleven patients (17%) were found to have mutations in MD-related genes, with COQ4 having the highest mutation rate owing to the Chinese-specific founder mutation (4/66, 6%). Twelve patients (12/66, 18%) had mutations in non-MD-related genes: ATP1A3 (n = 3, two were siblings), ALDH5A1, ARX, FA2H, KCNT1, LDHD, NEFL, NKX2-2, TBCK, and WAC. CONCLUSIONS: We confirmed that the COQ4:c.370G>A, p.(Gly124Ser) variant, was a founder mutation among the Southern Chinese population. Screening for this mutation should therefore be considered while diagnosing Chinese patients suspected to have MDs. Furthermore, WES has proven to be useful in detecting variants in patients suspected to have MDs because it helps to obtain an unbiased and precise genetic diagnosis for these diseases, which are genetically heterogeneous.

A fatal case of COQ7-associated primary coenzyme Q10 deficiency.

BACKGROUND: Primary coenzyme Q10 (CoQ10) deficiencies are clinically and genetically heterogeneous group of disorders associated with defects of genes involved in the CoQ10 biosynthesis pathway. COQ7-associated CoQ10 deficiency is very rare and only two cases have been reported. METHODS AND RESULTS: We report a patient with encephalo-myo-nephro-cardiopathy, persistent lactic acidosis, and basal ganglia lesions resulting in early infantile death. Using whole exome sequencing, we identified compound heterozygous variants in the COQ7 gene consisting of a deletion insertion resulting in frameshift [c.599_600delinsTAATGCATC, p.(Lys200Ilefs*56)] and a missense substitution [c.319C>T, p.(Arg107Trp), NM_016138.4]. Skin fibroblast studies showed decreased combined complex II + III activity and reduction in CoQ10 level. CONCLUSION: This third patient presenting with lethal encephalo-myo-nephro-cardiopathy represents the severe end of this ultra-rare mitochondrial disease caused by biallelic COQ7 mutations. The response to CoQ10 supplement is poor and alternative treatment strategies should be developed for a more effective management of this disorder.

ATP1A3 mutation in a Chinese girl with alternating hemiplegia of childhood--Potential target of treatment?

BACKGROUND: This Chinese girl had alternating hemiplegia of childhood (AHC) since 2 months. She failed to respond to anticonvulsants, antimigrainous drugs and calcium channel blockers but achieved complete remission steroid treatment for 4 weeks and relapsed after stopping steroid. PURPOSE: In order to clarify the unknown etiology, genetic analysis of ATP1A3 gene, which encodes the alpha3-subunit of the sodium/potassium-transporting ATPase (Na, K-ATPase), has been done by Sanger sequencing. RESULTS: A de novo heterozygous missense mutation (c.2401G>A; p.D801N) was identified in exon 17 of ATP1A3 gene and this is one of the hotspot mutations found in AHC patients. CONCLUSION: It will be interesting to further investigate whether Na, K-ATPase was the target of corticosteroid treatment.

SCN2A mutation in a Chinese boy with infantile spasm - response to Modified Atkins Diet.

BACKGROUND: Mutation of SCN2A, encoding for voltage-gated sodium channel type II alpha subunit, has been demonstrated in various epilepsy phenotypes, ranging from benign to severe epileptic disorders and recently this had been reported for cases with infantile spasm (IS). METHODS: We study a 6 years-old Chinese boy with severe developmental delay who had infantile spasm since 15 months. He later had severe intellectual disability and autistic features. He failed to respond to most anticonvulsants. Modified Atkins Diet was introduced at 4 years of age and he showed a seizure remission for 12 months with only 1 anticonvulsants. To clarify the unknown etiology, mutations were screened for genes associated with brain development or synaptic function. RESULTS: A heterozygous mutation (c.3631G>A; p.E1211K) was identified in exon 21 of SCN2A gene. This mutation has been reported previously only in a Japanese patient with IS. CONCLUSION: This is the first case of SCN2A mutation identified in Chinese. Similarity of our case and one Japanese case of infantile spasm indicated that this E1211K mutation is important as possible etiology of IS. Trial of Modified Atkins Diet for other cases of infantile spasm with similar SCN2A mutations is worthwhile pursuing.

Folinic acid responsive epilepsy in Ohtahara syndrome caused by STXBP1 mutation.

BACKGROUND: Ohtahara syndrome is a severe condition with early onset of recurrent unprovoked seizures associated with abnormal electroencephalography and global developmental delay. Folinic acid-responsive seizures are treatable causes of Ohtahara syndrome, which is thought to be due to recessive mutations in the ALDH7A1 gene, resulting in deficiency of antiquitin. METHOD: Here we report a girl with Ohtahara syndrome who exhibited transient folinic acid responsiveness but without evidence of antiquitin dysfunction. RESULTS: She was later found to have a known missense mutation (c.1439 C > T, p.P480 L) in exon 16 of the STXBP1 gene. CONCLUSION: For infants presenting with Ohtahara syndrome with responsiveness to folinic acid and negative antiquitin deficiency analyses, genetic testing for other possible causative genes such as STXBP1 mutation is recommended.

The importance of the olfactory rosettes in maintaining pituitary prolactin and prolactin-releasing peptide levels during hyposmotic acclimation in silver sea bream (Sparus sarba).

A potential role of the olfactory rosettes in maintaining prolactin (PRL) and prolactin-releasing peptide (PrRP) levels was examined in the euryhaline silver sea bream (Sparus sarba). The olfactory rosettes were surgically removed in silver sea bream adapted to hypo- (6 ppt) and hyper-osmotic (33 ppt) salinities and the mRNA expression of the two previously identified freshwater-adapting factors, prolactin (PRL) and prolactin-releasing peptide (PrRP), in silver sea bream was measured. The elevation of pituitary PRL and PrRP mRNA expression levels as seen in 6 ppt-adapted fish was abolished by surgical removal of the olfactory rosettes. The PRL and PrRP expression levels in fish adapted to 6 ppt were significantly lowered following olfactory rosette removal. On the other hand, hypothalamic PrRP mRNA expression in 6 ppt-adapted fish did not change. Specific signals for Na(+)-K(+)-ATPase but not CFTR mRNA expression were detected in the surface layers of olfactory epithelial cells by in situ hybridization. The mRNA abundance of CFTR and Na(+)-K(+)-ATPase α and ß subunits remained unchanged in the olfactory rosette of silver sea bream adapted to 0, 6, 12, 33 and 50 ppt for 4 weeks and in fish abruptly transferred from 33 ppt to 6 ppt. Data obtained from the olfactory rosette removal experiments suggest a possible role of the olfactory system for maintaining PRL and PrRP expression during hyposmotic acclimation in sea bream.

Effect of extracellular osmolality and ionic levels on pituitary prolactin release in euryhaline silver sea bream (Sparus sarba).

In many euryhaline fish, prolactin (PRL) plays a key role in freshwater adaptation. Consistent with this function, the present study showed a remarkable reduction in pituitary PRL content of silver sea bream abruptly transferred to low salinity (6ppt). This reduction in pituitary PRL content followed closely the temporal changes in serum osmolality and ion levels. Serum osmolality, Na(+) and Cl(-) levels of silver sea bream abruptly transferred to hyposmotic salinity (6ppt) were markedly reduced 2h after the transfer. The decline in pituitary PRL content lagged behind the serum changes implying that reduction in pituitary PRL content is a response to the drop in serum ion levels and osmotic pressure. Silver sea bream pituitary cells were dispersed and exposed to a medium with reduced ion levels and osmolality in vitro, and PRL released from pituitary cells was significantly elevated. In hyposmotic exposed anterior pituitary cells, cell volume exhibited a 20% increase when exposed to a medium with a 20% decrease in osmolality. The enlarged pituitary cells did not shrink until the surrounding hyposmotic medium was replaced, a phenomenon suggesting an osmosensing ability of silver sea bream PRL cells for PRL secretion in response to a change in extracellular osmotic pressure. The decrease in pituitary PRL content in vivo and stimulated pituitary PRL release in vitro under reduced osmolality together suggest hyposmotic exposure triggers PRL release from the pituitary.

Direct actions of cortisol, thyroxine and growth hormone on IGF-I mRNA expression in sea bream hepatocytes.

The present study aims to investigate potential regulatory effect of different growth-related hormones including growth hormone (GH), human insulin-like growth factor-I (hIGF-I), thyroxine (T(4)), triiodothyronine (T(3)) and cortisol, on insulin-like growth factor-I (IGF-I) mRNA expression of hepatocytes isolated from silver sea bream. By using real-time PCR, IGF-I mRNA expression profiles of hepatocytes in response to individual hormones were determined in vitro. Hepatocytes incubated with GH at concentrations of 10-1000 ng/mL showed significantly higher IGF-I expression, but the elevation was attenuated at high concentration of GH (1000 ng/mL). IGF-I expression remained unchanged in hepatocytes after incubation with hIGF-I. Hepatocytes incubated with T(4) at concentration of 1000 ng/mL exhibited a significant elevation in IGF-I expression, whereas no difference in IGF-I expression was demonstrated in hepatocytes after incubation with T(3). Upon incubation with cortisol (1-1000 ng/mL), IGF-I expression was significantly decreased in hepatocytes in a dose-dependent manner. Our study demonstrated that GH, T(4), and cortisol had direct modulatory effects on IGF-I expression in fish hepatocytes in vitro.

Prolactin-releasing peptide, a possible modulator of prolactin in the euryhaline silver sea bream (Sparus sarba): A molecular study.

PRL and PrRP cDNAs have been isolated from euryhaline silver sea bream (Sparus sarba). The PRL cDNA consists of 1360bp encoding 212 amino acids whereas the PrRP cDNA contains 631bp encoding preproPrRP with 122 amino acids. The mature PrRP sequence within the preprohormone is identical to the PrRPs isolated from other fish species. PRL mRNA was uniquely expressed in sea bream pituitary but PrRP mRNA was expressed in a variety of organs and tissues including the intestines, olfactory rosette and various brain regions such as hypothalamus and pituitary. Expression levels of PRL and PrRP mRNA have been examined in sea bream adapted to different salinities (0, 6, 12, 33 and 50ppt). In the pituitary, both PRL and PrRP mRNA were significantly higher in fish adapted to low salinities (0 and 6ppt) and the expression profiles of both hormones closely paralleled each other. However, expression of hypothalamic PrRP was significantly higher in fish adapted to iso-osmotic salinity (12ppt) when pituitary PRL expression was low. The present study demonstrates, for the first time, a synchronized mRNA expression pattern between PRL and PrRP in fish pituitary but a disparity of mRNA expression levels between hypothalamic PrRP and pituitary PRL during salinity adaptation. These data suggest that PrRP may possibly act as a local modulator in pituitary rather than a hypothalamic factor for regulation of pituitary PRL expression in silver sea bream.