Gabriela Oprea

Variants in BRSK2 , encoding brain specific kinase-2, have recently been associated with an autosomal dominant neurodevelopmental disorder (NDD). We have assembled 52 cases with heterozygous BRSK2 variants and variable neurodevelopmental phenotypes with frequent neuropsychiatric and behavioral symptoms. The variant spectrum included 15 different truncating variants, seven (potential) splice variants, three structural variants, and 12 different missense variants. Of the missense variants, seven were in the kinase domain, and the others in the UBA and the KA1 domain or outside domains. Variants occurred de novo in 19 cases and were inherited in 18. We utilized Drosophila melanogaster as a model and assessed viability and performed climbing and bang sensitivity assays upon knockdown of the fly orthologue sff or upon overexpression of wildtype or mutant human BRSK2 . Pan-neuronal knockdown of sff resulted in impaired locomotor behavior and seizure susceptibility. Ubiquitous or pan-neuronal overexpression of human wildtype BRSK2 in Drosophila resulted in lethality or locomotor impairment, respectively, indicating toxicity. Overexpressing mutant BRSK2 did not or incompletely affect viability and locomotor behavior for six of seven tested kinase domain missense variants and one KA1 domain variant, indicating a (partial) loss-of-function effect. Interestingly, overexpressing BRSK2 with the remaining missense variant from the kinase domain and the two most C-terminal missense variants resulted in possible gain of function. Our findings further delineate the clinical and molecular spectrum of BRSK2 -associated NDD and provide further insights into the role of BRSK2/sff in nervous system function and dysfunction.

Background The small nuclear RNA-activating complex polypeptide 4 (SNAPC4) encodes the largest scaffolding subunit of the SNAPc complex, essential for transcription of spliceosomal small nuclear RNAs (snRNAs) by RNA polymerase II and III. Although pathogenic variants in SNAPC4 have been associated with a rare neurodevelopmental disorder, the full phenotypic spectrum and underlying molecular mechanisms remain poorly defined. Methods We assembled an international cohort of 14 individuals harboring 18 distinct pathogenic SNAPC4 variants and one multi-exon deletion, three of which had been previously reported, including frameshift, splice-site, multi-exon deletions, and missense alleles. Detailed clinical and neuroimaging data were integrated with in silico structural and protein–protein interaction analyses to assess the impact of these variants on SNAPc architecture and function. Results SNAPC4 -related disease emerges as a highly heterogeneous clinical continuum rather than a uniform progressive disorder. Core features included global developmental delay and microcephaly, with variable trajectories ranging from static cognitive impairment to adult-onset progressive motor dysfunction, alongside neurobehavioral phenotypes such as autism and ADHD. Neuroimaging revealed abnormalities suggestive of impaired neurogenesis and gliogenesis, including lissencephaly-pachygyria and hypomyelinating leukodystrophy. Structural modeling supports a unifying loss-of-function (LoF) mechanism, whereby variants destabilize the SNAPc complex or disrupt key interfaces required for DNA binding and RNA polymerase recruitment, consistent with impaired SNAPc function and transcriptional dysregulation. Conclusion These findings redefine SNAPC4 -related disease as a stage-dependent neurodevelopmental spliceosomopathy, linking disruption of SNAPC4 to impaired SNAPc function and transcriptional dysregulation, with direct implications for diagnosis and variant interpretation.

Primary mitochondrial diseases (PMDs) affect approximately 1 in 4300 individuals and cause early-onset neuromuscular and multisystem dysfunction with reduced lifespan. They result from pathogenic variants in mitochondrial or nuclear DNA that impair oxidative phosphorylation. Cytochrome c oxidase (COX; complex IV) deficiency is a well-established cause of PMD, leading to a broad spectrum of phenotypes. COXFA4 (cytochrome c oxidase subunit FA4), formerly NDUFA4, is a nuclear-encoded COX subunit, but its role in disease remains poorly defined. We report the largest genetically confirmed cohort of COXFA4-related PMD to date, comprising 13 individuals from 12 families with biallelic pathogenic COXFA4 variants. All present with Leigh-like encephalopathy and complete loss of COXFA4 protein; however, patient-derived fibroblasts retain residual COX activity, with upregulation of COXFA4L2 (cytochrome c oxidase subunit FA4-like 2), a poorly characterised paralog. Here, we show that COXFA4 is a late-stage COX assembly subunit and identify a paralog-mediated compensatory mechanism with translational potential.

Autosomal recessive Limb-Girdle Muscular Dystrophy type R28 (LGMDR28; OMIM #620375) is one of the most recently identified subtypes of recessive LGMD. To date, 17 affected individuals from eight unrelated families have been reported to harbor biallelic variants in the HMGCR gene. Here, we report eleven individuals from six unrelated Middle Eastern families diagnosed with LGMDR28 and carrying homozygous variants in HMGCR . Our cohort recapitulated most of the previously described clinical features of the HMGCR-related phenotype. In addition, we also report a family with congenital onset of the condition as well as two affected individuals with liver failure. Through genome and exome sequencing, we identified three novel homozygous missense variants; c.2519G >A, p.(Arg840Gln), c.1784G >A, p.(Arg595His), and c.1783C>T, p.(Arg595Cys) and a homozygous in-frame deletion (c.1522_1524del, p.(Ser508del)) previously reported in compound heterozygous state with a second relevant variant and very recently as homozygous in an unrelated proband. This study consolidates the pathogenic role of HMGCR in LGMDR28 while expanding the phenotypic and mutations spectrum of LGMDR28.

The UNC13A gene encodes a presynaptic protein that is crucial for setting the strength and dynamics of information transfer between neurons. Here we describe a neurodevelopmental syndrome caused by germline coding or splice-site variants in UNC13A . The syndrome presents with variable degrees of developmental delay and intellectual disability, seizures of different types, tremor and dyskinetic movements and, in some cases, death in early childhood. Using assays with expression of UNC13A variants in mouse hippocampal neurons and in Caenorhabditis elegans , we identify three mechanisms of pathogenicity, including reduction in synaptic strength caused by reduced UNC13A protein expression, increased neurotransmission caused by UNC13A gain-of-function and impaired regulation of neurotransmission by second messenger signalling. Based on a strong genotype–phenotype-functional correlation, we classify three UNC13A syndrome subtypes (types A–C). We conclude that the precise regulation of neurotransmitter release by UNC13A is critical for human nervous system function.

Background/Objectives: Although biallelic pathogenic variants in different DNAH gene family members have been associated with infertility, the role of DNAH12 in this disorder is still incompletely understood. To date, few patients have been shown to have infertility due to biallelic variants in this gene. Here, we report two more unrelated patients with infertility who carry homozygous variants in DNAH12. Methods: This study included two male patients with primary infertility and oligoasthenoteratozoospermia (OAT). Patient 1 was a 32-year-old with 1.5 years of infertility and no chronic illnesses or prior assisted reproductive technologies (ARTs). Patient 2 was a 49-year-old with 24 years of infertility, a history of varicocelectomy, and the occasional use of PRN analgesics for bone pain. Using genome sequencing, we identified two homozygous variants: c.3757C>A, p. Pro1253Thr, and c.11086-1G>A, p.?, in patients 1 and 2, respectively. Results: Our findings add supportive evidence that DNAH12 is a gene implicated in rare cases of male infertility. The identification of these homozygous variants in two additional patients supports the association between DNAH12 variants and reproductive dysfunction. Conclusions: This study highlights the need for further research on the role of DNAH12, including functional studies to clarify the mechanisms contributing to infertility.

Myogenic fusion, primarily regulated by the Myomaker and Myomixer proteins, is essential for skeletal muscle development, yet its mechanisms remain poorly understood. This study presents the clinical and molecular details of the third and fourth reported patients with biallelic variants in MYMX, the gene that encodes Myomixer. We identified a homozygous truncating variant [c.107 T > A (p.Leu36Ter)] and a homozygous stop-codon loss variant [c.255 A > G (p.Ter85TrpextTer41)] in MYMX, both associated with a complex neuromuscular syndrome characterized by generalized hypotonia, congenital myopathy, facial nerve palsy, growth restriction and facial dysmorphism. Additional variable features include hearing loss (confirmed in one patient, suspected in the other), scoliosis, joint contractures, cleft palate, hypoglossia, potentially contributing to Pierre Robin sequence, and abnormalities on neuroimaging studies including cerebellar atrophy and Chiari 1 deformity. Comparative analysis of patients with pathogenic variants in MYMK and MYMX, including our cases, reveals largely overlapping phenotypes, underscoring their synergistic role in myofiber formation and implicating their involvement in the etiology of neuromuscular conditions.

Background Studies in the field of genomic secondary findings (SF) are diverse regarding participants’ characteristics; sequencing methods; versions of the ACMG SF gene list. Aim and methods Based on whole genome sequencing (WGS) and version 3.1 of ACMG SF list (ACMG SF), we studied SF in 863 individuals from Pakistan: 62% males; 80% had consanguineous parents. In addition to the ACMG SF we have generated a list of gene-disease pairs that have a clear epidemiological and medically actionable value (non-ACMG SF) in Pakistan. Results The total rate of SF was 4.6%, with rates of ACMG SF – 2.7% and non-AGMG SF – 1.9%. 75.0% of ACMG SF were related to cardiovascular diseases (CVD); cancer predisposition syndromes accounted for 16.7%. Among non-ACMG SF 18.8% belong to eye diseases, followed by neuromuscular – 12.5%, metabolic – 12.5%, and urinary system diseases – 12.5%; CVD accounted for 6.3%. We found high proportion of biallelic mutations among both ACMG (4.2%) and non-ACMG (50%) SF. Conclusions The frequency of ACMG SF is within the range reported in most studies. High proportion of CVD can be explained by inclusion of additional CVD in the ACMG v3.1 SF list. 1.9% of non-ACMG SF and high proportion of biallelic variants are relevant to epidemiology of Pakistan as a country with high rate of consanguineous marriages. In such countries the ACMG criteria for SF can be expanded, and our list of non-ACMG SF is one example. Our findings may help guide the development of standards of practice in genomic medicine and drive future research.

Rare denovo variants represent a significant cause of neurodevelopmental delay and intellectual disability (ID). Exome sequencing was performed on 4351 patients with global developmental delay, seizures, microcephaly, macrocephaly, motor delay, delayed speech and language development, or ID according to Human Phenotype Ontology (HPO) terms. All patients had previously undergone whole exome sequencing as part of diagnostic genetic testing with a focus on variants in genes implicated in neurodevelopmental disorders up to January 2017. This resulted in a genetic diagnosis in 1336 of the patients. In this study, we specifically searched for variants in 14 recently implicated novel neurodevelopmental disorder (NDD) genes. We identified 65 rare, protein-changing variants in 11 of these 14 novel candidate genes. Fourteen variants in CDK13, CHD4, KCNQ3, KMT5B, TCF20, and ZBTB18 were scored pathogenic or likely pathogenic. Of note, two of these patients had a previously identified cause of their disease, and thus, multiple molecular diagnoses were made including pathogenic/likely pathogenic variants in FOXG1 and CDK13 or in TMEM237 and KMT5B. Looking for pathogenic variants in newly identified NDD genes enabled us to provide a molecular diagnosis to 14 patients and their close relatives and caregivers. This underlines the relevance of re-evaluation of existing exome data on a regular basis to improve the diagnostic yield and serve the needs of our patients.