NODAL variants are associated with a continuum of laterality defects from simple D-transposition of the great arteries to heterotaxy.

BACKGROUND: NODAL signaling plays a critical role in embryonic patterning and heart development in vertebrates. Genetic variants resulting in perturbations of the TGF-ß/NODAL signaling pathway have reproducibly been shown to cause laterality defects in humans. To further explore this association and improve genetic diagnosis, the study aims to identify and characterize a broader range of NODAL variants in a large number of individuals with laterality defects. METHODS: We re-analyzed a cohort of 321 proband-only exomes of individuals with clinically diagnosed laterality congenital heart disease (CHD) using family-based, rare variant genomic analyses. To this cohort we added 12 affected subjects with known NODAL variants and CHD from institutional research and clinical cohorts to investigate an allelic series. For those with candidate contributory variants, variant allele confirmation and segregation analysis were studied by Sanger sequencing in available family members. Array comparative genomic hybridization and droplet digital PCR were utilized for copy number variants (CNV) validation and characterization. We performed Human Phenotype Ontology (HPO)-based quantitative phenotypic analyses to dissect allele-specific phenotypic differences. RESULTS: Missense, nonsense, splice site, indels, and/or structural variants of NODAL were identified as potential causes of heterotaxy and other laterality defects in 33 CHD cases. We describe a recurrent complex indel variant for which the nucleic acid secondary structure predictions implicate secondary structure mutagenesis as a possible mechanism for formation. We identified two CNV deletion alleles spanning NODAL in two unrelated CHD cases. Furthermore, 17 CHD individuals were found (16/17 with known Hispanic ancestry) to have the c.778G > A:p.G260R NODAL missense variant which we propose reclassification from variant of uncertain significance (VUS) to likely pathogenic. Quantitative HPO-based analyses of the observed clinical phenotype for all cases with p.G260R variation, including heterozygous, homozygous, and compound heterozygous cases, reveal clustering of individuals with biallelic variation. This finding provides evidence for a genotypic-phenotypic correlation and an allele-specific gene dosage model. CONCLUSION: Our data further support a role for rare deleterious variants in NODAL as a cause for sporadic human laterality defects, expand the repertoire of observed anatomical complexity of potential cardiovascular anomalies, and implicate an allele specific gene dosage model.

Genomic Balancing Act: Deciphering DNA rearrangements in the Complex Chromosomal Aberration involving 5p15.2, 2q31.1 and 18q21.32.

Despite extensive research into the genetic underpinnings of neurodevelopmental disorders (NDD), many clinical cases remain unresolved. We studied a female proband with a NDD, mildly dysmorphic facial features, and brain stem hypoplasia on neuroimaging. Comprehensive genomic analyses revealed a terminal 5p loss and terminal 18q gain in the proband while a diploid copy number for chromosomes 5 and 18 in both parents. Genomic investigations in the proband identified an unbalanced translocation t(5;18) with additional genetic material from chromosome 2 (2q31.3) inserted at the breakpoint, pointing to a complex chromosomal rearrangement (CCR) involving 5p15.2, 2q31.3, and 18q21.32. Breakpoint junction analyses enabled by long read genome sequencing unveiled the presence of four distinct junctions in the father, who is carrier of a balanced CCR. The proband inherited from the father both the abnormal chromosome 5 resulting in segmental aneusomies of chr5 (loss) and chr18 (gain) and a der(2) homologue. Evidences suggest a chromoplexy mechanism for this CCR derivation, involving double-strand breaks (DSBs) repaired by non-homologous end joining (NHEJ) or alternative end joining (alt-EJ). The complexity of the CCR and the segregation of homologues elucidate the genetic model for this family. This study demonstrates the importance of combining multiple genomic technologies to uncover genetic causes of complex neurodevelopmental syndrome and to better understand genetic disease mechanisms.

Biallelic variation in the choline and ethanolamine transporter FLVCR1 underlies a pleiotropic disease spectrum from adult neurodegeneration to severe developmental disorders.

FLVCR1 encodes Feline leukemia virus subgroup C receptor 1 (FLVCR1), a solute carrier (SLC) transporter within the Major Facilitator Superfamily. FLVCR1 is a widely expressed transmembrane protein with plasma membrane and mitochondrial isoforms implicated in heme, choline, and ethanolamine transport. While Flvcr1 knockout mice die in utero with skeletal malformations and defective erythropoiesis reminiscent of Diamond-Blackfan anemia, rare biallelic pathogenic FLVCR1 variants are linked to childhood or adult-onset neurodegeneration of the retina, spinal cord, and peripheral nervous system. We ascertained from research and clinical exome sequencing 27 individuals from 20 unrelated families with biallelic ultra-rare missense and predicted loss-of-function (pLoF) FLVCR1 variant alleles. We characterize an expansive FLVCR1 phenotypic spectrum ranging from adult-onset retinitis pigmentosa to severe developmental disorders with microcephaly, reduced brain volume, epilepsy, spasticity, and premature death. The most severely affected individuals, including three individuals with homozygous pLoF variants, share traits with Flvcr1 knockout mice and Diamond-Blackfan anemia including macrocytic anemia and congenital skeletal malformations. Pathogenic FLVCR1 missense variants primarily lie within transmembrane domains and reduce choline and ethanolamine transport activity compared with wild-type FLVCR1 with minimal impact on FLVCR1 stability or subcellular localization. Several variants disrupt splicing in a mini-gene assay which may contribute to genotype-phenotype correlations. Taken together, these data support an allele-specific gene dosage model in which phenotypic severity reflects residual FLVCR1 activity. This study expands our understanding of Mendelian disorders of choline and ethanolamine transport and demonstrates the importance of choline and ethanolamine in neurodevelopment and neuronal homeostasis.

HMZDupFinder: a robust computational approach for detecting intragenic homozygous duplications from exome sequencing data.

Homozygous duplications contribute to genetic disease by altering gene dosage or disrupting gene regulation and can be more deleterious to organismal biology than heterozygous duplications. Intragenic exonic duplications can result in loss-of-function (LoF) or gain-of-function (GoF) alleles that when homozygosed, i.e. brought to homozygous state at a locus by identity by descent or state, could potentially result in autosomal recessive (AR) rare disease traits. However, the detection and functional interpretation of homozygous duplications from exome sequencing data remains a challenge. We developed a framework algorithm, HMZDupFinder, that is designed to detect exonic homozygous duplications from exome sequencing (ES) data. The HMZDupFinder algorithm can efficiently process large datasets and accurately identifies small intragenic duplications, including those associated with rare disease traits. HMZDupFinder called 965 homozygous duplications with three or less exons from 8,707 ES with a recall rate of 70.9% and a precision of 16.1%. We experimentally confirmed 8/10 rare homozygous duplications. Pathogenicity assessment of these copy number variant alleles allowed clinical genomics contextualization for three homozygous duplications alleles, including two affecting known OMIM disease genes EDAR (MIM# 224900), TNNT1(MIM# 605355), and one variant in a novel candidate disease gene: PAAF1.

Break-induced replication underlies formation of inverted triplications and generates unexpected diversity in haplotype structures.

Background: The duplication-triplication/inverted-duplication (DUP-TRP/INV-DUP) structure is a type of complex genomic rearrangement (CGR) hypothesized to result from replicative repair of DNA due to replication fork collapse. It is often mediated by a pair of inverted low-copy repeats (LCR) followed by iterative template switches resulting in at least two breakpoint junctions in cis . Although it has been identified as an important mutation signature of pathogenicity for genomic disorders and cancer genomes, its architecture remains unresolved and is predicted to display at least four structural variation (SV) haplotypes. Results: Here we studied the genomic architecture of DUP-TRP/INV-DUP by investigating the genomic DNA of 24 patients with neurodevelopmental disorders identified by array comparative genomic hybridization (aCGH) on whom we found evidence for the existence of 4 out of 4 predicted SV haplotypes. Using a combination of short-read genome sequencing (GS), long- read GS, optical genome mapping and StrandSeq the haplotype structure was resolved in 18 samples. This approach refined the point of template switching between inverted LCRs in 4 samples revealing a DNA segment of ∼2.2-5.5 kb of 100% nucleotide similarity. A prediction model was developed to infer the LCR used to mediate the non-allelic homology repair. Conclusions: These data provide experimental evidence supporting the hypothesis that inverted LCRs act as a recombinant substrate in replication-based repair mechanisms. Such inverted repeats are particularly relevant for formation of copy-number associated inversions, including the DUP-TRP/INV-DUP structures. Moreover, this type of CGR can result in multiple conformers which contributes to generate diverse SV haplotypes in susceptible loci .

Biallelic missense variants in COG3 cause a congenital disorder of glycosylation with impairment of retrograde vesicular trafficking.

Biallelic variants in genes for seven out of eight subunits of the conserved oligomeric Golgi complex (COG) are known to cause recessive congenital disorders of glycosylation (CDG) with variable clinical manifestations. COG3 encodes a constituent subunit of the COG complex that has not been associated with disease traits in humans. Herein, we report two COG3 homozygous missense variants in four individuals from two unrelated consanguineous families that co-segregated with COG3-CDG presentations. Clinical phenotypes of affected individuals include global developmental delay, severe intellectual disability, microcephaly, epilepsy, facial dysmorphism, and variable neurological findings. Biochemical analysis of serum transferrin from one family showed the loss of a single sialic acid. Western blotting on patient-derived fibroblasts revealed reduced COG3 and COG4. Further experiments showed delayed retrograde vesicular recycling in patient cells. This report adds to the knowledge of the COG-CDG network by providing collective evidence for a COG3-CDG rare disease trait and implicating a likely pathology of the disorder as the perturbation of Golgi trafficking.

Monoallelic variation in DHX9, the gene encoding the DExH-box helicase DHX9, underlies neurodevelopment disorders and Charcot-Marie-Tooth disease.

DExD/H-box RNA helicases (DDX/DHX) are encoded by a large paralogous gene family; in a subset of these human helicase genes, pathogenic variation causes neurodevelopmental disorder (NDD) traits and cancer. DHX9 encodes a BRCA1-interacting nuclear helicase regulating transcription, R-loops, and homologous recombination and exhibits the highest mutational constraint of all DDX/DHX paralogs but remains unassociated with disease traits in OMIM. Using exome sequencing and family-based rare-variant analyses, we identified 20 individuals with de novo, ultra-rare, heterozygous missense or loss-of-function (LoF) DHX9 variant alleles. Phenotypes ranged from NDDs to the distal symmetric polyneuropathy axonal Charcot-Marie-Tooth disease (CMT2). Quantitative Human Phenotype Ontology (HPO) analysis demonstrated genotype-phenotype correlations with LoF variants causing mild NDD phenotypes and nuclear localization signal (NLS) missense variants causing severe NDD. We investigated DHX9 variant-associated cellular phenotypes in human cell lines. Whereas wild-type DHX9 was restricted to the nucleus, NLS missense variants abnormally accumulated in the cytoplasm. Fibroblasts from an individual with an NLS variant also showed abnormal cytoplasmic DHX9 accumulation. CMT2-associated missense variants caused aberrant nucleolar DHX9 accumulation, a phenomenon previously associated with cellular stress. Two NDD-associated variants, p.Gly411Glu and p.Arg761Gln, altered DHX9 ATPase activity. The severe NDD-associated variant p.Arg141Gln did not affect DHX9 localization but instead increased R-loop levels and double-stranded DNA breaks. Dhx9-/- mice exhibited hypoactivity in novel environments, tremor, and sensorineural hearing loss. All together, these results establish DHX9 as a critical regulator of mammalian neurodevelopment and neuronal homeostasis.

Rare variant enrichment analysis supports GREB1L as a contributory driver gene in the etiology of Mayer-Rokitansky-Küster-Hauser syndrome.

Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is characterized by aplasia of the female reproductive tract; the syndrome can include renal anomalies, absence or dysgenesis, and skeletal anomalies. While functional models have elucidated several candidate genes, only WNT4 (MIM: 603490) variants have been definitively associated with a subtype of MRKH with hyperandrogenism (MIM: 158330). DNA from 148 clinically diagnosed MRKH probands across 144 unrelated families and available family members from North America, Europe, and South America were exome sequenced (ES) and by family-based genomics analyzed for rare likely deleterious variants. A replication cohort consisting of 442 Han Chinese individuals with MRKH was used to further reproduce GREB1L findings in diverse genetic backgrounds. Proband and OMIM phenotypes annotated using the Human Phenotype Ontology were analyzed to quantitatively delineate the phenotypic spectrum associated with GREB1L variant alleles found in our MRKH cohort and those previously published. This study reports 18 novel GREB1L variant alleles, 16 within a multiethnic MRKH cohort and two within a congenital scoliosis cohort. Cohort-wide analyses for a burden of rare variants within a single gene identified likely damaging variants in GREB1L (MIM: 617782), a known disease gene for renal hypoplasia and uterine abnormalities (MIM: 617805), in 16 of 590 MRKH probands. GREB1L variant alleles, including a CNV null allele, were found in 8 MRKH type 1 probands and 8 MRKH type II probands. This study used quantitative phenotypic analyses in a worldwide multiethnic cohort to identify and strengthen the association of GREB1L to isolated uterine agenesis (MRKH type I) and syndromic MRKH type II.

A biallelic frameshift indel in PPP1R35 as a cause of primary microcephaly.

Protein phosphatase 1 regulatory subunit 35 (PPP1R35) encodes a centrosomal protein required for recruiting microtubule-binding elongation machinery. Several proteins in this centriole biogenesis pathway correspond to established primary microcephaly (MCPH) genes, and multiple model organism studies hypothesize PPP1R35 as a candidate MCPH gene. Here, using exome sequencing (ES) and family-based rare variant analyses, we report a homozygous, frameshifting indel deleting the canonical stop codon in the last exon of PPP1R35 [Chr7: c.753_*3delGGAAGCGTAGACCinsCG (p.Trp251Cysfs*22)]; the variant allele maps in a 3.7 Mb block of absence of heterozygosity (AOH) in a proband with severe MCPH (-4.3 SD at birth, -6.1 SD by 42 months), pachygyria, and global developmental delay from a consanguineous Turkish kindred. Droplet digital PCR (ddPCR) confirmed mutant mRNA expression in fibroblasts. In silico prediction of the translation of mutant PPP1R35 is expected to be elongated by 18 amino acids before encountering a downstream stop codon. This complex indel allele is absent in public databases (ClinVar, gnomAD, ARIC, 1000 genomes) and our in-house database of 14,000+ exomes including 1800+ Turkish exomes supporting predicted pathogenicity. Comprehensive literature searches for PPP1R35 variants yielded two probands affected with severe microcephaly (-15 SD and -12 SD) with the same homozygous indel from a single, consanguineous, Iranian family from a cohort of 404 predominantly Iranian families. The lack of heterozygous cases in two large cohorts representative of the genetic background of these two families decreased our suspicion of a founder allele and supports the contention of a recurrent mutation. We propose two potential secondary structure mutagenesis models for the origin of this variant allele mediated by hairpin formation between complementary GC rich segments flanking the stop codon via secondary structure mutagenesis.

TCEAL1 loss-of-function results in an X-linked dominant neurodevelopmental syndrome and drives the neurological disease trait in Xq22.2 deletions.

An Xq22.2 region upstream of PLP1 has been proposed to underly a neurological disease trait when deleted in 46,XX females. Deletion mapping revealed that heterozygous deletions encompassing the smallest region of overlap (SRO) spanning six Xq22.2 genes (BEX3, RAB40A, TCEAL4, TCEAL3, TCEAL1, and MORF4L2) associate with an early-onset neurological disease trait (EONDT) consisting of hypotonia, intellectual disability, neurobehavioral abnormalities, and dysmorphic facial features. None of the genes within the SRO have been associated with monogenic disease in OMIM. Through local and international collaborations facilitated by GeneMatcher and Matchmaker Exchange, we have identified and herein report seven de novo variants involving TCEAL1 in seven unrelated families: three hemizygous truncating alleles; one hemizygous missense allele; one heterozygous TCEAL1 full gene deletion; one heterozygous contiguous deletion of TCEAL1, TCEAL3, and TCEAL4; and one heterozygous frameshift variant allele. Variants were identified through exome or genome sequencing with trio analysis or through chromosomal microarray. Comparison with previously reported Xq22 deletions encompassing TCEAL1 identified a more-defined syndrome consisting of hypotonia, abnormal gait, developmental delay/intellectual disability especially affecting expressive language, autistic-like behavior, and mildly dysmorphic facial features. Additional features include strabismus, refractive errors, variable nystagmus, gastroesophageal reflux, constipation, dysmotility, recurrent infections, seizures, and structural brain anomalies. An additional maternally inherited hemizygous missense allele of uncertain significance was identified in a male with hypertonia and spasticity without syndromic features. These data provide evidence that TCEAL1 loss of function causes a neurological rare disease trait involving significant neurological impairment with features overlapping the EONDT phenotype in females with the Xq22 deletion.

The multiple de novo copy number variant (MdnCNV) phenomenon presents with peri-zygotic DNA mutational signatures and multilocus pathogenic variation.

BACKGROUND: The multiple de novo copy number variant (MdnCNV) phenotype is described by having four or more constitutional de novo CNVs (dnCNVs) arising independently throughout the human genome within one generation. It is a rare peri-zygotic mutational event, previously reported to be seen once in every 12,000 individuals referred for genome-wide chromosomal microarray analysis due to congenital abnormalities. These rare families provide a unique opportunity to understand the genetic factors of peri-zygotic genome instability and the impact of dnCNV on human diseases. METHODS: Chromosomal microarray analysis (CMA), array-based comparative genomic hybridization, short- and long-read genome sequencing (GS) were performed on the newly identified MdnCNV family to identify de novo mutations including dnCNVs, de novo single-nucleotide variants (dnSNVs), and indels. Short-read GS was performed on four previously published MdnCNV families for dnSNV analysis. Trio-based rare variant analysis was performed on the newly identified individual and four previously published MdnCNV families to identify potential genetic etiologies contributing to the peri-zygotic genomic instability. Lin semantic similarity scores informed quantitative human phenotype ontology analysis on three MdnCNV families to identify gene(s) driving or contributing to the clinical phenotype. RESULTS: In the newly identified MdnCNV case, we revealed eight de novo tandem duplications, each ~ 1 Mb, with microhomology at 6/8 breakpoint junctions. Enrichment of de novo single-nucleotide variants (SNV; 6/79) and de novo indels (1/12) was found within 4 Mb of the dnCNV genomic regions. An elevated post-zygotic SNV mutation rate was observed in MdnCNV families. Maternal rare variant analyses identified three genes in distinct families that may contribute to the MdnCNV phenomenon. Phenotype analysis suggests that gene(s) within dnCNV regions contribute to the observed proband phenotype in 3/3 cases. CNVs in two cases, a contiguous gene duplication encompassing PMP22 and RAI1 and another duplication affecting NSD1 and SMARCC2, contribute to the clinically observed phenotypic manifestations. CONCLUSIONS: Characteristic features of dnCNVs reported here are consistent with a microhomology-mediated break-induced replication (MMBIR)-driven mechanism during the peri-zygotic period. Maternal genetic variants in DNA repair genes potentially contribute to peri-zygotic genomic instability. Variable phenotypic features were observed across a cohort of three MdnCNV probands, and computational quantitative phenotyping revealed that two out of three had evidence for the contribution of more than one genetic locus to the proband's phenotype supporting the hypothesis of de novo multilocus pathogenic variation (MPV) in those families.

A reverse genetics and genomics approach to gene paralog function and disease: Myokymia and the juxtaparanode.

The leucine-rich glioma-inactivated (LGI) family consists of four highly conserved paralogous genes, LGI1-4, that are highly expressed in mammalian central and/or peripheral nervous systems. LGI1 antibodies are detected in subjects with autoimmune limbic encephalitis and peripheral nerve hyperexcitability syndromes (PNHSs) such as Isaacs and Morvan syndromes. Pathogenic variations of LGI1 and LGI4 are associated with neurological disorders as disease traits including familial temporal lobe epilepsy and neurogenic arthrogryposis multiplex congenita 1 with myelin defects, respectively. No human disease has been reported associated with either LGI2 or LGI3. We implemented exome sequencing and family-based genomics to identify individuals with deleterious variants in LGI3 and utilized GeneMatcher to connect practitioners and researchers worldwide to investigate the clinical and electrophysiological phenotype in affected subjects. We also generated Lgi3-null mice and performed peripheral nerve dissection and immunohistochemistry to examine the juxtaparanode LGI3 microarchitecture. As a result, we identified 16 individuals from eight unrelated families with loss-of-function (LoF) bi-allelic variants in LGI3. Deep phenotypic characterization showed LGI3 LoF causes a potentially clinically recognizable PNHS trait characterized by global developmental delay, intellectual disability, distal deformities with diminished reflexes, visible facial myokymia, and distinctive electromyographic features suggestive of motor nerve instability. Lgi3-null mice showed reduced and mis-localized Kv1 channel complexes in myelinated peripheral axons. Our data demonstrate bi-allelic LoF variants in LGI3 cause a clinically distinguishable disease trait of PNHS, most likely caused by disturbed Kv1 channel distribution in the absence of LGI3.

Developmental genomics of limb malformations: Allelic series in association with gene dosage effects contribute to the clinical variability.

Genetic heterogeneity, reduced penetrance, and variable expressivity, the latter including asymmetric body axis plane presentations, have all been described in families with congenital limb malformations (CLMs). Interfamilial and intrafamilial heterogeneity highlight the complexity of the underlying genetic pathogenesis of these developmental anomalies. Family-based genomics by exome sequencing (ES) and rare variant analyses combined with whole-genome array-based comparative genomic hybridization were implemented to investigate 18 families with limb birth defects. Eleven of 18 (61%) families revealed explanatory variants, including 7 single-nucleotide variant alleles and 3 copy number variants (CNVs), at previously reported "disease trait associated loci": BHLHA9, GLI3, HOXD cluster, HOXD13, NPR2, and WNT10B. Breakpoint junction analyses for all three CNV alleles revealed mutational signatures consistent with microhomology-mediated break-induced replication, a mechanism facilitated by Alu/Alu-mediated rearrangement. Homozygous duplication of BHLHA9 was observed in one Turkish kindred and represents a novel contributory genetic mechanism to Gollop-Wolfgang Complex (MIM: 228250), where triplication of the locus has been reported in one family from Japan (i.e., 4n = 2n + 2n versus 4n = 3n + 1n allelic configurations). Genes acting on limb patterning are sensitive to a gene dosage effect and are often associated with an allelic series. We extend an allele-specific gene dosage model to potentially assist, in an adjuvant way, interpretations of interconnections among an allelic series, clinical severity, and reduced penetrance of the BHLHA9-related CLM spectrum.

Biallelic Variants in the Ectonucleotidase ENTPD1 Cause a Complex Neurodevelopmental Disorder with Intellectual Disability, Distinct White Matter Abnormalities, and Spastic Paraplegia.

OBJECTIVE: Human genomics established that pathogenic variation in diverse genes can underlie a single disorder. For example, hereditary spastic paraplegia is associated with >80 genes, with frequently only few affected individuals described for each gene. Herein, we characterize a large cohort of individuals with biallelic variation in ENTPD1, a gene previously linked to spastic paraplegia 64 (Mendelian Inheritance in Man # 615683). METHODS: Individuals with biallelic ENTPD1 variants were recruited worldwide. Deep phenotyping and molecular characterization were performed. RESULTS: A total of 27 individuals from 17 unrelated families were studied; additional phenotypic information was collected from published cases. Twelve novel pathogenic ENTPD1 variants are described (NM 001776.6): c.398_399delinsAA; p.(Gly133Glu), c.540del; p.(Thr181Leufs*18), c.640del; p.(Gly216Glufs*75), c.185 T > G; p.(Leu62*), c.1531 T > C; p.(*511Glnext*100), c.967C > T; p.(Gln323*), c.414-2_414-1del, and c.146 A > G; p.(Tyr49Cys) including 4 recurrent variants c.1109 T > A; p.(Leu370*), c.574-6_574-3del, c.770_771del; p.(Gly257Glufs*18), and c.1041del; p.(Ile348Phefs*19). Shared disease traits include childhood onset, progressive spastic paraplegia, intellectual disability (ID), dysarthria, and white matter abnormalities. In vitro assays demonstrate that ENTPD1 expression and function are impaired and that c.574-6_574-3del causes exon skipping. Global metabolomics demonstrate ENTPD1 deficiency leads to impaired nucleotide, lipid, and energy metabolism. INTERPRETATION: The ENTPD1 locus trait consists of childhood disease onset, ID, progressive spastic paraparesis, dysarthria, dysmorphisms, and white matter abnormalities, with some individuals showing neurocognitive regression. Investigation of an allelic series of ENTPD1 (1) expands previously described features of ENTPD1-related neurological disease, (2) highlights the importance of genotype-driven deep phenotyping, (3) documents the need for global collaborative efforts to characterize rare autosomal recessive disease traits, and (4) provides insights into disease trait neurobiology. ANN NEUROL 2022;92:304-321.

Novel RETREG1 (FAM134B) founder allele is linked to HSAN2B and renal disease in a Turkish family.

Hereditary sensory and autonomic neuropathy type 2B (HSAN2B) is a rare autosomal recessive peripheral neuropathy caused by biallelic variants in RETREG1 (formerly FAM134B). HSAN2B is characterized by sensory impairment resulting in skin ulcerations, amputations, and osteomyelitis as well as variable weakness, spasticity, and autonomic dysfunction. Here, we report four affected individuals with recurrent osteomyelitis, ulceration, and amputation of hands and feet, sensory neuropathy, hyperhidrosis, urinary incontinence, and renal failure from a family without any known shared parental ancestry. Due to the history of chronic recurrent multifocal osteomyelitis and microcytic anemia, a diagnosis of Majeed syndrome was considered; however, sequencing of LPIN2 was negative. Family-based exome sequencing (ES) revealed a novel homozygous ultrarare RETREG1 variant NM_001034850.2:c.321G>A;p.Trp107Ter. Electrophysiological studies of the proband demonstrated axonal sensorimotor neuropathy predominantly in the lower extremities. Consistent with the lack of shared ancestry, the coefficient of inbreeding calculated from ES data was low (F = 0.002), but absence of heterozygosity (AOH) analysis demonstrated a 7.2 Mb AOH block surrounding the variant consistent with a founder allele. Two of the four affected individuals had unexplained renal failure which has not been reported in HSAN2B cases to date. Therefore, this report describes a novel RETREG1 founder allele and suggests renal failure may be an unrecognized feature of the RETREG1-disease spectrum.

De novo variants in EMC1 lead to neurodevelopmental delay and cerebellar degeneration and affect glial function in Drosophila.

BACKGROUND: The endoplasmic reticulum (ER)-membrane protein complex (EMC) is a multi-protein transmembrane complex composed of 10 subunits that functions as a membrane-protein chaperone. Variants in EMC1 lead to neurodevelopmental delay and cerebellar degeneration. Multiple families with biallelic variants have been published, yet to date, only a single report of a monoallelic variant has been described, and functional evidence is sparse. METHODS: Exome sequencing was used to investigate the genetic cause underlying severe developmental delay in three unrelated children. EMC1 variants were modeled in Drosophila, using loss-of-function (LoF) and overexpression studies. Glial-specific and neuronal-specific assays were used to determine whether the dysfunction was specific to one cell type. RESULTS: Exome sequencing identified de novo variants in EMC1 in three individuals affected by global developmental delay, hypotonia, seizures, visual impairment and cerebellar atrophy. All variants were located at Pro582 or Pro584. Drosophila studies indicated that imbalance of EMC1-either overexpression or knockdown-results in pupal lethality and suggest that the tested homologous variants are LoF alleles. In addition, glia-specific gene dosage, overexpression or knockdown, of EMC1 led to lethality, whereas neuron-specific alterations were tolerated. DISCUSSION: We establish de novo monoallelic EMC1 variants as causative of a neurological disease trait by providing functional evidence in a Drosophila model. The identified variants failed to rescue the lethality of a null allele. Variations in dosage of the wild-type EMC1, specifically in glia, lead to pupal lethality, which we hypothesize results from the altered stoichiometry of the multi-subunit protein complex EMC.

Biallelic variants in SLC38A3 encoding a glutamine transporter cause epileptic encephalopathy.

The solute carrier (SLC) superfamily encompasses >400 transmembrane transporters involved in the exchange of amino acids, nutrients, ions, metals, neurotransmitters and metabolites across biological membranes. SLCs are highly expressed in the mammalian brain; defects in nearly 100 unique SLC-encoding genes (OMIM: https://www.omim.org) are associated with rare Mendelian disorders including developmental and epileptic encephalopathy and severe neurodevelopmental disorders. Exome sequencing and family-based rare variant analyses on a cohort with neurodevelopmental disorders identified two siblings with developmental and epileptic encephalopathy and a shared deleterious homozygous splicing variant in SLC38A3. The gene encodes SNAT3, a sodium-coupled neutral amino acid transporter and a principal transporter of the amino acids asparagine, histidine, and glutamine, the latter being the precursor for the neurotransmitters GABA and glutamate. Additional subjects with a similar developmental and epileptic encephalopathy phenotype and biallelic predicted-damaging SLC38A3 variants were ascertained through GeneMatcher and collaborations with research and clinical molecular diagnostic laboratories. Untargeted metabolomic analysis was performed to identify novel metabolic biomarkers. Ten individuals from seven unrelated families from six different countries with deleterious biallelic variants in SLC38A3 were identified. Global developmental delay, intellectual disability, hypotonia, and absent speech were common features while microcephaly, epilepsy, and visual impairment were present in the majority. Epilepsy was drug-resistant in half. Metabolomic analysis revealed perturbations of glutamate, histidine, and nitrogen metabolism in plasma, urine, and CSF of selected subjects, potentially representing biomarkers of disease. Our data support the contention that SLC38A3 is a novel disease gene for developmental and epileptic encephalopathy and illuminate the likely pathophysiology of the disease as perturbations in glutamine homeostasis.

Quantitative dissection of multilocus pathogenic variation in an Egyptian infant with severe neurodevelopmental disorder resulting from multiple molecular diagnoses.

Genomic sequencing and clinical genomics have demonstrated that substantial subsets of atypical and/or severe disease presentations result from multilocus pathogenic variation (MPV) causing blended phenotypes. In an infant with a severe neurodevelopmental disorder, four distinct molecular diagnoses were found by exome sequencing (ES). The blended phenotype that includes brain malformation, dysmorphism, and hypotonia was dissected using the Human Phenotype Ontology (HPO). ES revealed variants in CAPN3 (c.259C > G:p.L87V), MUSK (c.1781C > T:p.A594V), NAV2 (c.1996G > A:p.G666R), and ZC4H2 (c.595A > C:p.N199H). CAPN3, MUSK, and ZC4H2 are established disease genes linked to limb-girdle muscular dystrophy (OMIM# 253600), congenital myasthenia (OMIM# 616325), and Wieacker-Wolff syndrome (WWS; OMIM# 314580), respectively. NAV2 is a retinoic-acid responsive novel disease gene candidate with biological roles in neurite outgrowth and cerebellar dysgenesis in mouse models. Using semantic similarity, we show that no gene identified by ES individually explains the proband phenotype, but rather the totality of the clinically observed disease is explained by the combination of disease-contributing effects of the identified genes. These data reveal that multilocus pathogenic variation can result in a blended phenotype with each gene affecting a different part of the nervous system and nervous system-muscle connection. We provide evidence from this n = 1 study that in patients with MPV and complex blended phenotypes resulting from multiple molecular diagnoses, quantitative HPO analysis can allow for dissection of phenotypic contribution of both established disease genes and novel disease gene candidates not yet proven to cause human disease.

High prevalence of multilocus pathogenic variation in neurodevelopmental disorders in the Turkish population.

Neurodevelopmental disorders (NDDs) are clinically and genetically heterogenous; many such disorders are secondary to perturbation in brain development and/or function. The prevalence of NDDs is > 3%, resulting in significant sociocultural and economic challenges to society. With recent advances in family-based genomics, rare-variant analyses, and further exploration of the Clan Genomics hypothesis, there has been a logarithmic explosion in neurogenetic "disease-associated genes" molecular etiology and biology of NDDs; however, the majority of NDDs remain molecularly undiagnosed. We applied genome-wide screening technologies, including exome sequencing (ES) and whole-genome sequencing (WGS), to identify the molecular etiology of 234 newly enrolled subjects and 20 previously unsolved Turkish NDD families. In 176 of the 234 studied families (75.2%), a plausible and genetically parsimonious molecular etiology was identified. Out of 176 solved families, deleterious variants were identified in 218 distinct genes, further documenting the enormous genetic heterogeneity and diverse perturbations in human biology underlying NDDs. We propose 86 candidate disease-trait-associated genes for an NDD phenotype. Importantly, on the basis of objective and internally established variant prioritization criteria, we identified 51 families (51/176 = 28.9%) with multilocus pathogenic variation (MPV), mostly driven by runs of homozygosity (ROHs) - reflecting genomic segments/haplotypes that are identical-by-descent. Furthermore, with the use of additional bioinformatic tools and expansion of ES to additional family members, we established a molecular diagnosis in 5 out of 20 families (25%) who remained undiagnosed in our previously studied NDD cohort emanating from Turkey.