Refining the 9q34.3 microduplication syndrome reveals mild neurodevelopmental features associated with a distinct global DNA methylation profile.

Precise regulation of gene expression is important for correct neurodevelopment. 9q34.3 deletions affecting the EHMT1 gene result in a syndromic neurodevelopmental disorder named Kleefstra syndrome. In contrast, duplications of the 9q34.3 locus encompassing EHMT1 have been suggested to cause developmental disorders, but only limited information has been available. We have identified 15 individuals from 10 unrelated families, with 9q34.3 duplications <1.5 Mb in size, encompassing EHMT1 entirely. Clinical features included mild developmental delay, mild intellectual disability or learning problems, autism spectrum disorder, and behavior problems. The individuals did not consistently display dysmorphic features, congenital anomalies, or growth abnormalities. DNA methylation analysis revealed a weak DNAm profile for the cases with 9q34.3 duplication encompassing EHMT1, which could segregate the majority of the affected cases from controls. This study shows that individuals with 9q34.3 duplications including EHMT1 gene present with mild non-syndromic neurodevelopmental disorders and DNA methylation changes different from Kleefstra syndrome.

CAMTA1-related disorder: Phenotypic and molecular characterization of 26 new individuals and literature review.

Calmodulin-binding transcriptional activator 1 (CAMTA1) is highly expressed in the brain and plays a role in cell cycle regulation, cell differentiation, regulation of long-term memory, and initial development, maturation, and survival of cerebellar neurons. The existence of human neurological phenotypes, including cerebellar dysfunction with variable cognitive and behavioral abnormalities (CECBA), associated with CAMTA1 variants, has further supported its role in brain functions. In this study, we phenotypically and molecularly characterize the largest cohort of individuals (n = 26) with 23 novel CAMTA1 variants (frameshift-7, nonsense-6, splicing-1, initiation codon-1, missense-5, and intragenic deletions-3) and compare the findings with all previously reported cases (total = 53). We show that the most notable phenotypic findings are developmental delay/intellectual disability, unsteady or uncoordinated gait, hypotonia, behavioral problems, and eye abnormalities. In addition, there is a high incidence of dysarthria, dysgraphia, microcephaly, gastrointestinal abnormalities, sleep difficulties, and nonspecific brain MRI findings; a few of which have been under-reported. More than one third of the variants in this cohort were inherited from an asymptomatic or mildly affected parent suggesting reduced penetrance and variable expressivity. Our cohort provides a comprehensive characterization of the spectrum of phenotypes and genotypes among individuals with CECBA and the large data will facilitate counseling and formulating management plans and surveillance recommendations for these individuals.

Improving diagnostic precision in primary ovarian insufficiency using comprehensive genetic and autoantibody testing.

STUDY QUESTION: Is it possible to find the cause of primary ovarian insufficiency (POI) in more women by extensive screening? SUMMARY ANSWER: Adding next generation sequencing techniques including a POI-associated gene panel, extended whole exome sequencing data, as well as specific autoantibody assays to the recommended diagnostic investigations increased the determination of a potential etiological diagnosis of POI from 11% to 41%. WHAT IS KNOWN ALREADY: POI affects ∼1% of women. Clinical presentations and pathogenic mechanisms are heterogeneous and include genetic, autoimmune, and environmental factors, but the underlying etiology remains unknown in the majority of cases. STUDY DESIGN, SIZE, DURATION: Prospective cross-sectional study of 100 women with newly diagnosed POI of unknown cause consecutively referred to Haukeland University Hospital, Bergen, Norway, January 2019 to December 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: In addition to standard recommended diagnostic investigations including screening for chromosomal anomalies and premutations in the fragile X mental retardation 1 gene (FMR1) we used whole exome sequencing, including targeted analysis of 103 ovarian-related genes, and assays of autoantibodies against steroid cell antigens. MAIN RESULTS AND THE ROLE OF CHANCE: We identified chromosomal aberrations in 8%, FMR1 premutations in 3%, genetic variants related to POI in 16%, and autoimmune POI in 3%. Furthermore in 11% we identified POI associated genetic Variants of unknown signifcance (VUS). A homozygous pathogenic variant in the ZSWIM7 gene (NM_001042697.2) was found in two women, corroborating this as a novel cause of monogenic POI. No associations between phenotypes and genotypes were found. LIMITATIONS, REASONS FOR CAUTION: Use of candidate genetic and autoimmune markers limit the possibility to discover new markers. To further investigate the genetic variants, family studies would have been useful. We found a relatively high proportion of genetic variants in women from Africa and lack of genetic diversity in the genomic databases can impact diagnostic accuracy. WIDER IMPLICATIONS OF THE FINDINGS: Since no specific clinical or biochemical markers predicted the underlying cause of POI discussion of which tests should be part of diagnostic screening in clinical practice remains open. New technology has altered the availability and effectiveness of genetic testing, and cost-effectiveness analyses are required to aid sustainable diagnostics. STUDY FUNDING/COMPETING INTEREST(S): The study was supported by grants and fellowships from Stiftelsen Kristian Gerhard Jebsen, the Novonordisk Foundation, the Norwegian Research Council, University of Bergen, and the Regional Health Authorities of Western Norway. The authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: NCT04082169.

Börjeson-Forssman-Lehmann syndrome: delineating the clinical and allelic spectrum in 14 new families.

Börjeson-Forssman-Lehmann syndrome (BFLS) is an X-linked intellectual disability syndrome caused by variants in the PHF6 gene. We ascertained 19 individuals from 15 families with likely pathogenic or pathogenic PHF6 variants (11 males and 8 females). One family had previously been reported. Six variants were novel. We analysed the clinical and genetic findings in our series and compared them with reported BFLS patients. Affected males had classic features of BFLS including intellectual disability, distinctive facies, large ears, gynaecomastia, hypogonadism and truncal obesity. Carrier female relatives of affected males were unaffected or had only mild symptoms. The phenotype of affected females with de novo variants overlapped with the males but included linear skin hyperpigmentation and a higher frequency of dental, retinal and cortical brain anomalies. Complications observed in our series included keloid scarring, digital fibromas, absent vaginal orifice, neuropathy, umbilical hernias, and talipes. Our analysis highlighted sex-specific differences in PHF6 variant types and locations. Affected males often have missense variants or small in-frame deletions while affected females tend to have truncating variants or large deletions/duplications. Missense variants were found in a minority of affected females and clustered in the highly constrained PHD2 domain of PHF6. We propose recommendations for the evaluation and management of BFLS patients. These results further delineate and extend the genetic and phenotypic spectrum of BFLS.

The spectrum of pyridoxine dependent epilepsy across the age span: A nationwide retrospective observational study.

BACKGROUND: Pyridoxine-dependent epilepsy (PDE) is a rare seizure disorder usually presenting with neonatal seizures. Most cases are caused by biallelic pathogenic ALDH7A1variants. While anti-seizure medications are ineffective, pyridoxine provides seizure control, and dietary interventions may be of benefit. As the natural history beyond adolescence is insufficiently explored, our study aimed to assess the spectrum of PDE at various ages in Norway. METHODS: Patients were ascertained by contacting all Norwegian paediatric, neurological, and neurohabilitation departments and relevant professional societies. Medical records were collected and reviewed. RESULTS: We identified 15 patients treated for PDE; 13 had ALDH7A1 variants (PDE-ALDH7A1), one had PNPO deficiency, and in one, aetiology remained obscure. Of those with PDE-ALDH7A1, 12 were alive at time of study; five were > 18 years old and six were < 4 years. Median age was 10 years (range 2 months-53 years). Estimated minimum prevalence was 6.3/million among children and 1.2/million among adults. Ten had seizure onset on the first day of life. Perinatal complications and neuroradiological abnormalities suggested additional seizure aetiologies in several patients. Pyridoxine had immediate effect in six, while six had delayed (>1 h) or uncertain effect. Median delay from first seizure to continuous treatment was 11 days (range 0-42). Nine experienced breakthrough seizures with intercurrent disease or due to pyridoxine discontinuation. Cognitive outcomes ranged from normal to severe intellectual disability. The condition appeared to remain stable in adult life. SIGNIFICANCE: We found a much higher prevalence of PDE-ALDH7A1 in children relative to adults, suggesting previous underdiagnosis and early mortality. Perinatal complications are common and can delay diagnosis and initiation of pyridoxine treatment. Lifelong and continuous treatment with pyridoxine is imperative. Due to better diagnostics and survival, the number of adult patients is expected to rise.

Possible association of trichorhinophalangeal syndrome I and intracranial subependymoma.

Trichorhinophalangeal syndromes (TRPSs) are rare hereditary syndromes with autosomal dominant inheritance. Patients exhibit abnormalities including bulbous pear-shaped nose, broad columella, and long and flat philtrum, fine, sparse, brittle, slow-growing scalp hair, skeletal abnormalities, and short stature. Three families; age at subependymoma surgery, pathogenic TRPS1(NM_014112.5) variant, and subependymoma number are described.

Fat Malabsorption and Ursodeoxycholic Acid Treatment in Children With Reduced Organic Solute Transporter-α (SLC51A) Expression.

Objectives: A bile acid homeostasis disorder was suspected in 2 siblings and their second cousin who presented in infancy with fat malabsorption, severe fat-soluble vitamin deficiency, rickets, and mild liver involvement. Our aims were to identify the genetic cause, describe the disease, and evaluate the response to ursodeoxycholic acid (UDCA) treatment. Methods: Whole exome sequencing, immunohistochemistry of duodenal biopsies and candidate variant testing in a cell-based model was performed. Fecal fat excretion, serum bile acids, 7α-hydroxy-4-cholesten-3-one (C4), and fibroblast growth factor 19 (FGF19) were quantified in both siblings on and off UDCA treatment. Results: A novel homozygous variant of SLC51A, which encodes the bile acid carrier organic solute transporter (OST)-α, was identified in all affected children. OSTα protein expression was readily detected by immunohistochemistry in duodenum of pediatric control subjects but not in the affected siblings. The siblings had low serum levels of bile acids and C4 and high serum levels of FGF19 consistent with repression of hepatic bile acid synthesis. On treatment with UDCA, fecal fat excretion was reduced and serum levels of C4, FGF19, and liver enzymes normalized. Conclusions: We report an apparent deficiency of OSTα associated with early onset fat malabsorption and mild liver involvement. The clinical presentation partially overlaps previous reports for 3 patients with OSTα or OSTß deficiency and extends the clinical spectrum associated with loss of SLC51A expression. Our data suggest that repression of hepatic bile acid synthesis contributes to fat malabsorption in OSTα-OSTß deficiency but can be partly reversed with UDCA treatment.

Further characterization of Borjeson-Forssman-Lehmann syndrome in females due to de novo variants in PHF6.

While inherited hemizygous variants in PHF6 cause X-linked recessive Borjeson-Forssman-Lehmann syndrome (BFLS) in males, de novo heterozygous variants in females are associated with an overlapping but distinct phenotype, including moderate to severe intellectual disability, characteristic facial dysmorphism, dental, finger and toe anomalies, and linear skin pigmentation. By personal communication with colleagues, we assembled 11 additional females with BFLS due to variants in PHF6. We confirm the distinct phenotype to include variable intellectual disability, recognizable facial dysmorphism and other anomalies. We observed skewed X-inactivation in blood and streaky skin pigmentation compatible with functional mosaicism. Variants occurred de novo in 10 individuals, of whom one was only mildly affected and transmitted it to her more severely affected daughter. The mutational spectrum comprises a two-exon deletion, five truncating, one splice-site and three missense variants, the latter all located in the PHD2 domain and predicted to severely destabilize the domain structure. This observation supports the hypothesis of more severe variants in females contributing to gender-specific phenotypes in addition to or in combination with effects of X-inactivation and functional mosaicism. Therefore, our findings further delineate the clinical and mutational spectrum of female BFLS and provide further insights into possible genotype-phenotype correlations between females and males.

LRFN5 locus structure is associated with autism and influenced by the sex of the individual and locus conversions.

LRFN5 is a regulator of synaptic development and the only gene in a 5.4 Mb mammalian-specific conserved topologically associating domain (TAD); the LRFN5 locus. An association between locus structural changes and developmental delay (DD) and/or autism was suggested by several cases in DECIPHER and own records. More significantly, we found that maternal inheritance of a specific LRFN5 locus haplotype segregated with an identical type of autism in distantly related males. This autism-susceptibility haplotype had a specific TAD pattern. We also found a male/female quantitative difference in the amount histone-3-lysine-9-associated chromatin around the LRFN5 gene itself (p < 0.01), possibly related to the male-restricted autism susceptibility. To better understand locus behavior, the prevalence of a 60 kb deletion polymorphism was investigated. Surprisingly, in three cohorts of individuals with DD (n = 8757), the number of deletion heterozygotes was 20%-26% lower than expected from Hardy-Weinberg equilibrium. This suggests allelic interaction, also because the conversions from heterozygosity to wild-type or deletion homozygosity were of equal magnitudes. Remarkably, in a control group of medical students (n = 1416), such conversions were three times more common (p = 0.00001), suggesting a regulatory role of this allelic interaction. Taken together, LRFN5 regulation appears unusually complex, and LRFN5 dysregulation could be an epigenetic cause of autism. LAY SUMMARY: LRFN5 is involved with communication between brain cells. The gene sits alone in a huge genomic niche, called the LRFN5 locus, of complex structure and high mammalian conservation. We have found that a specific locus structure increases autism susceptibility in males, but we do not yet know how common this epigenetic cause of autism is. It is, however, a cause that potentially could explain why higher-functioning autism is more common in males than females.

Further delineation of the clinical spectrum of White-Sutton syndrome: 12 new individuals and a review of the literature.

White-Sutton syndrome (WHSUS) is a neurodevelopmental disorder caused by heterozygous loss-of-function variants in POGZ. Through the Deciphering Developmental Disorders study and clinical testing, we identified 12 individuals from 10 families with pathogenic or likely pathogenic variants in POGZ (eight de novo and two inherited). Most individuals had delayed development and/or intellectual disability. We analyzed the clinical findings in our series and combined it with data from 89 previously reported individuals. The results demonstrate WHSUS is associated with variable developmental delay or intellectual disability, increased risk of obesity, visual defects, craniofacial dysmorphism, sensorineural hearing loss, feeding problems, seizures, and structural brain malformations. Our series includes further individuals with rod-cone dystrophy, cleft lip and palate, congenital diaphragmatic hernia, and duplicated renal drainage system, suggesting these are rare complications of WHSUS. In addition, we describe an individual with a novel, de novo missense variant in POGZ and features of WHSUS. Our work further delineates the phenotypic spectrum of WHSUS highlighting the variable severity of this disorder and the observation of familial pathogenic POGZ variants.

Deep exploration of a CDKN1C mutation causing a mixture of Beckwith-Wiedemann and IMAGe syndromes revealed a novel transcript associated with developmental delay.

BACKGROUND: Loss-of-function mutations in CDKN1C cause overgrowth, that is, Beckwith-Wiedemann syndrome (BWS), while gain-of-function variants in the gene's PCNA binding motif cause a growth-restricted condition called IMAGe syndrome. We report on a boy with a remarkable mixture of both syndromes, with developmental delay and microcephaly as additional features. METHODS: Whole-exome DNA sequencing and ultra-deep RNA sequencing of leucocyte-derived and fibroblast-derived mRNA were performed in the family. RESULTS: We found a maternally inherited variant in the IMAGe hotspot region: NM_000076.2(CDKN1C) c.822_826delinsGAGCTG. The asymptomatic mother had inherited this variant from her mosaic father with mild BWS features. This delins caused tissue-specific frameshifting resulting in at least three novel mRNA transcripts in the boy. First, a splice product causing CDKN1C truncation was the likely cause of BWS. Second, an alternative splice product in fibroblasts encoded IMAGe-associated amino acid substitutions. Third, we speculate that developmental delay is caused by a change in the alternative CDKN1C-201 (ENST00000380725.1) transcript, encoding a novel isoform we call D (UniProtKB: A6NK88). Isoform D is distinguished from isoforms A and B by alternative splicing within exon 1 that changes the reading frame of the last coding exon. Remarkably, this delins changed the reading frame back to the isoform A/B type, resulting in a hybrid D-A/B isoform. CONCLUSION: Three different cell-type-dependent RNA products can explain the co-occurrence of both BWS and IMAGe features in the boy. Possibly, brain expression of hybrid isoform D-A/B is the cause of developmental delay and microcephaly, a phenotypic feature not previously reported in CDKN1C patients.

Truncating and zinc-finger variants in GLI2 are associated with hypopituitarism.

Variants in transcription factor GLI2 have been associated with hypopituitarism and structural brain abnormalities, occasionally including holoprosencephaly (HPE). Substantial phenotypic variability and nonpenetrance have been described, posing difficulties in the counseling of affected families. We present three individuals with novel likely pathogenic GLI2 variants, two with truncating and one with a de novo missense variant p.(Ser548Leu), and review the literature for comprehensive phenotypic descriptions of individuals with confirmed pathogenic (a) intragenic GLI2 variants and (b) chromosome 2q14.2 deletions encompassing only GLI2. We show that most of the 31 missense variants previously reported as pathogenic are likely benign or, at most, low-risk variants. Four Zn-finger variants: p.(Arg479Gly), p.(Arg516Pro), p.(Gly518Lys), and p.(Tyr575His) were classified as likely pathogenic, and three other variants as possibly pathogenic: p.(Pro253Ser), p.(Ala593Val), and p.(Pro1243Leu). We analyze the phenotypic descriptions of 60 individuals with pathogenic GLI2 variants and evidence a morbidity spectrum that includes hypopituitarism (58%), HPE (6%) or other brain structure abnormalities (15%), orofacial clefting (17%) and dysmorphic facial features (35%). We establish that truncating and Zn-finger variants in GLI2 are associated with a high risk of hypopituitarism, and that a solitary median maxillary central incisor is part of the GLI2-related phenotypic variability. The most prevalent phenotypic feature is post-axial polydactyly (65%) which is also the mildest phenotypic expression of the condition, reported in many parents of individuals with systemic findings. Our approach clarifies clinical risks and the important messages to discuss in counseling for a pathogenic GLI2 variant.

The blended phenotype of a germline RIT1 and a mosaic PIK3CA variant.

We report a patient with a germline RIT1 and a mosaic PIK3CA variant. The diagnosis of the RASopathy was confirmed by targeted sequencing following the identification of transient cardiomyopathy in a patient with PIK3CA-related overgrowth spectrum (PROS). Our observation confirms that the PIK3CA gain-of-function (GoF) variant effects dominate those of the RASopathy, and the resulting blended phenotype mostly resembles megalencephaly-capillary malformation syndrome (MCAP PROS). There appears to be interaction between RIT1 and PI3K-AKT because the latter pathway is needed for the growth-promoting activity of the first, at least in adenocarcinomas, but the details of this interaction are not known. If so, the PIK3CA somatic variant may not be just a chance event. It could also be of etiological relevance that Rit activation mediates resistance to cellular stress-that is, promotes cell survival. This anti-apoptotic effect could also make it more likely that a cell that spontaneously acquires a PIK3CA GoF variant will survive and proliferate. We aim to encourage clinicians to investigate atypical findings in individuals with PROS. If further similar cases are reported, this would suggest that the establishment of PROS mosaicism is facilitated by the background of a RASopathy.

Double paternal uniparental isodisomy 7 and 15 presenting with Beckwith-Wiedemann spectrum features.

Here we describe for the first time double paternal uniparental isodisomy (iUPD) 7 and 15 in a baby boy with features in the Beckwith-Wiedemann syndrome spectrum (BWSp) (placentomegaly, hyperinsulinism, enlarged viscera, hemangiomas, and earlobe creases) in addition to conjugated hyperbilirubinemia. His phenotype was also reminiscent of genome-wide paternal uniparental isodisomy. We discuss the most likely origin of the UPDs: a maternal double monosomy 7 and 15 rescued by duplication of the paternal chromosomes after fertilization. So far, paternal UPD7 is not associated with an abnormal phenotype, whereas paternal UPD15 causes Angelman syndrome. Methylation analysis for other clinically relevant imprinting disorders, including BWSp, was normal. Therefore, we hypothesized that the double UPD affected other imprinted genes. To look for such effects, patient fibroblast RNA was isolated and analyzed for differential expression compared to six controls. We did not find apparent transcription differences in imprinted genes outside Chromosomes 7 and 15 in patient fibroblast. PEG10 (7q21.3) was the only paternally imprinted gene on these chromosomes up-regulated beyond double-dose expectation (sixfold). We speculate that a high PEG10 level could have a growth-promoting effect as his phenotype was not related to aberrations in BWS locus on 11p15.5 after DNA, RNA, and methylation testing. However, many genes in gene sets associated with growth were up-regulated. This case broadens the phenotypic spectrum of UPDs but does not show evidence of involvement of an imprinted gene network.

Mutations in EPHB4 cause human venous valve aplasia.

Venous valve (VV) failure causes chronic venous insufficiency, but the molecular regulation of valve development is poorly understood. A primary lymphatic anomaly, caused by mutations in the receptor tyrosine kinase EPHB4, was recently described, with these patients also presenting with venous insufficiency. Whether the venous anomalies are the result of an effect on VVs is not known. VV formation requires complex "organization" of valve-forming endothelial cells, including their reorientation perpendicular to the direction of blood flow. Using quantitative ultrasound, we identified substantial VV aplasia and deep venous reflux in patients with mutations in EPHB4. We used a GFP reporter in mice to study expression of its ligand, ephrinB2, and analyzed developmental phenotypes after conditional deletion of floxed Ephb4 and Efnb2 alleles. EphB4 and ephrinB2 expression patterns were dynamically regulated around organizing valve-forming cells. Efnb2 deletion disrupted the normal endothelial expression patterns of the gap junction proteins connexin37 and connexin43 (both required for normal valve development) around reorientating valve-forming cells and produced deficient valve-forming cell elongation, reorientation, polarity, and proliferation. Ephb4 was also required for valve-forming cell organization and subsequent growth of the valve leaflets. These results uncover a potentially novel cause of primary human VV aplasia.

Genetic Dominant Variants in STUB1, Segregating in Families with SCA48, Display In Vitro Functional Impairments Indistinctive from Recessive Variants Associated with SCAR16.

Variants in STUB1 cause both autosomal recessive (SCAR16) and dominant (SCA48) spinocerebellar ataxia. Reports from 18 STUB1 variants causing SCA48 show that the clinical picture includes later-onset ataxia with a cerebellar cognitive affective syndrome and varying clinical overlap with SCAR16. However, little is known about the molecular properties of dominant STUB1 variants. Here, we describe three SCA48 families with novel, dominantly inherited STUB1 variants (p.Arg51_Ile53delinsProAla, p.Lys143_Trp147del, and p.Gly249Val). All the patients developed symptoms from 30 years of age or later, all had cerebellar atrophy, and 4 had cognitive/psychiatric phenotypes. Investigation of the structural and functional consequences of the recombinant C-terminus of HSC70-interacting protein (CHIP) variants was performed in vitro using ubiquitin ligase activity assay, circular dichroism assay and native polyacrylamide gel electrophoresis. These studies revealed that dominantly and recessively inherited STUB1 variants showed similar biochemical defects, including impaired ubiquitin ligase activity and altered oligomerization properties of the CHIP. Our findings expand the molecular understanding of SCA48 but also mean that assumptions concerning unaffected carriers of recessive STUB1 variants in SCAR16 families must be re-evaluated. More investigations are needed to verify the disease status of SCAR16 heterozygotes and elucidate the molecular relationship between SCA48 and SCAR16 diseases.

Genotype-phenotype correlations and novel molecular insights into the DHX30-associated neurodevelopmental disorders.

BACKGROUND: We aimed to define the clinical and variant spectrum and to provide novel molecular insights into the DHX30-associated neurodevelopmental disorder. METHODS: Clinical and genetic data from affected individuals were collected through Facebook-based family support group, GeneMatcher, and our network of collaborators. We investigated the impact of novel missense variants with respect to ATPase and helicase activity, stress granule (SG) formation, global translation, and their effect on embryonic development in zebrafish. SG formation was additionally analyzed in CRISPR/Cas9-mediated DHX30-deficient HEK293T and zebrafish models, along with in vivo behavioral assays. RESULTS: We identified 25 previously unreported individuals, ten of whom carry novel variants, two of which are recurrent, and provide evidence of gonadal mosaicism in one family. All 19 individuals harboring heterozygous missense variants within helicase core motifs (HCMs) have global developmental delay, intellectual disability, severe speech impairment, and gait abnormalities. These variants impair the ATPase and helicase activity of DHX30, trigger SG formation, interfere with global translation, and cause developmental defects in a zebrafish model. Notably, 4 individuals harboring heterozygous variants resulting either in haploinsufficiency or truncated proteins presented with a milder clinical course, similar to an individual harboring a de novo mosaic HCM missense variant. Functionally, we established DHX30 as an ATP-dependent RNA helicase and as an evolutionary conserved factor in SG assembly. Based on the clinical course, the variant location, and type we establish two distinct clinical subtypes. DHX30 loss-of-function variants cause a milder phenotype whereas a severe phenotype is caused by HCM missense variants that, in addition to the loss of ATPase and helicase activity, lead to a detrimental gain-of-function with respect to SG formation. Behavioral characterization of dhx30-deficient zebrafish revealed altered sleep-wake activity and social interaction, partially resembling the human phenotype. CONCLUSIONS: Our study highlights the usefulness of social media to define novel Mendelian disorders and exemplifies how functional analyses accompanied by clinical and genetic findings can define clinically distinct subtypes for ultra-rare disorders. Such approaches require close interdisciplinary collaboration between families/legal representatives of the affected individuals, clinicians, molecular genetics diagnostic laboratories, and research laboratories.

Bi-allelic KARS1 pathogenic variants affecting functions of cytosolic and mitochondrial isoforms are associated with a progressive and multisystem disease.

KARS1 encodes a lysyl-transfer RNA synthetase (LysRS) that links lysine to its cognate transfer RNA. Two different KARS1 isoforms exert functional effects in cytosol and mitochondria. Bi-allelic pathogenic variants in KARS1 have been associated to sensorineural hearing and visual loss, neuropathy, seizures, and leukodystrophy. We report the clinical, biochemical, and neuroradiological features of nine individuals with KARS1-related disorder carrying 12 different variants with nine of them being novel. The consequences of these variants on the cytosol and/or mitochondrial LysRS were functionally validated in yeast mutants. Most cases presented with severe neurological features including congenital and progressive microcephaly, seizures, developmental delay/intellectual disability, and cerebral atrophy. Oculo-motor dysfunction and immuno-hematological problems were present in six and three cases, respectively. A yeast growth defect of variable severity was detected for most variants on both cytosolic and mitochondrial isoforms. The detrimental effects of two variants on yeast growth were partially rescued by lysine supplementation. Congenital progressive microcephaly, oculo-motor dysfunction, and immuno-hematological problems are emerging phenotypes in KARS1-related disorder. The data in yeast emphasize the role of both mitochondrial and cytosolic isoforms in the pathogenesis of KARS1-related disorder and supports the therapeutic potential of lysine supplementation at least in a subset of patients.