Variable Intrafamilial Expression of ABCB4 Disease.

Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a rare cholestatic liver disease with autosomal recessive inheritance caused by mutations in the ABCB4 gene. The clinical presentation of PFIC3 varies significantly, displaying incomplete penetrance without clear genotype-phenotype correlations. As such, the suitability of living-related liver donation for children with advanced disease has been questioned. We report here the long-term follow-up of a patient with PFIC3 resulting in decompensated cirrhosis at 11 years who successfully underwent living donor liver transplantation from his father, who carried the same ABCB4 homozygous mutation.

Recurrent de novo missense variants across multiple histone H4 genes underlie a neurodevelopmental syndrome.

Chromatin is essentially an array of nucleosomes, each of which consists of the DNA double-stranded fiber wrapped around a histone octamer. This organization supports cellular processes such as DNA replication, DNA transcription, and DNA repair in all eukaryotes. Human histone H4 is encoded by fourteen canonical histone H4 genes, all differing at the nucleotide level but encoding an invariant protein. Here, we present a cohort of 29 subjects with de novo missense variants in six H4 genes (H4C3, H4C4, H4C5, H4C6, H4C9, and H4C11) identified by whole-exome sequencing and matchmaking. All individuals present with neurodevelopmental features of intellectual disability and motor and/or gross developmental delay, while non-neurological features are more variable. Ten amino acids are affected, six recurrently, and are all located within the H4 core or C-terminal tail. These variants cluster to specific regions of the core H4 globular domain, where protein-protein interactions occur with either other histone subunits or histone chaperones. Functional consequences of the identified variants were evaluated in zebrafish embryos, which displayed abnormal general development, defective head organs, and reduced body axis length, providing compelling evidence for the causality of the reported disorder(s). While multiple developmental syndromes have been linked to chromatin-associated factors, missense-bearing histone variants (e.g., H3 oncohistones) are only recently emerging as a major cause of pathogenicity. Our findings establish a broader involvement of H4 variants in developmental syndromes.

A homozygous duplication of the FGG exon 8-intron 8 junction causes congenital afibrinogenemia. Lessons learned from the study of a large consanguineous Turkish family.

Congenital afibrinogenemia is the most severe congenital fibrinogen disorder, characterized by undetectable fibrinogen in circulation. Causative mutations can be divided into two main classes: null mutations with no protein production at all and missense mutations producing abnormal protein chains that are retained inside the cell. The vast majority of cases are due to single base pair mutations or small insertions or deletions in the coding regions or intron-exon junctions of FGB, FGA and FGG. Only a few large rearrangements have been described, all deletions involving FGA. Here we report the characterization of a 403 bp duplication of the FGG exon 8-intron 8 junction accounting for congenital afibrinogenemia in a large consanguineous family from Turkey. This mutation, which had escaped detection by Sanger sequencing of short polymerase chain reaction (PCR) amplicons of coding sequences and splice sites, was identified by studying multiple alignments of reads obtained from whole exome sequencing of a heterozygous individual followed by PCR amplification and sequencing of a larger portion of FGG. Because the mutation duplicates the donor splice site of intron 8, we predicted that the impact of the mutation would be on FGG transcript splicing. Analysis of mRNA produced by cells transiently transfected with normal or mutant minigene constructs showed that the duplication causes production of several aberrant FGG transcripts generating premature truncating codons.

Benefits of Exome Sequencing in Children with Suspected Isolated Hearing Loss.

PURPOSE: Hearing loss is characterized by an extensive genetic heterogeneity and remains a common disorder in children. Molecular diagnosis is of particular benefit in children, and permits the early identification of clinically-unrecognized hearing loss syndromes, which permits effective clinical management and follow-up, including genetic counselling. METHODS: We performed whole-exome sequencing with the analysis of a panel of 189 genes associated with hearing loss in a prospective cohort of 61 children and 9 adults presenting mainly with isolated hearing loss. RESULTS: The overall diagnostic rate using exome sequencing was 47.2% (52.5% in children; 22% in adults). In children with confirmed molecular results, 17/32 (53.2%) showed autosomal recessive inheritance patterns, 14/32 (43.75%) showed an autosomal dominant condition, and one case had X-linked hearing loss. In adults, the two patients showed an autosomal dominant inheritance pattern. Among the 32 children, 17 (53.1%) had nonsyndromic hearing loss and 15 (46.7%) had syndromic hearing loss. One adult was diagnosed with syndromic hearing loss and one with nonsyndromic hearing loss. The most common causative genes were STRC (5 cases), GJB2 (3 cases), COL11A1 (3 cases), and ACTG1 (3 cases). CONCLUSIONS: Exome sequencing has a high diagnostic yield in children with hearing loss and can reveal a syndromic hearing loss form before other organs/systems become involved, allowing the surveillance of unrecognized present and/or future complications associated with these syndromes.

Truncating variants in the SHANK1 gene are associated with a spectrum of neurodevelopmental disorders.

PURPOSE: In this study, we aimed to characterize the clinical phenotype of a SHANK1-related disorder and define the functional consequences of SHANK1 truncating variants. METHODS: Exome sequencing (ES) was performed for six individuals who presented with neurodevelopmental disorders. Individuals were ascertained with the use of GeneMatcher and Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources (DECIPHER). We evaluated potential nonsense-mediated decay (NMD) of two variants by making knock-in cell lines of endogenous truncated SHANK1, and expressed the truncated SHANK1 complementary DNA (cDNA) in HEK293 cells and cultured hippocampal neurons to examine the proteins. RESULTS: ES detected de novo truncating variants in SHANK1 in six individuals. Evaluation of NMD resulted in stable transcripts, and the truncated SHANK1 completely lost binding with Homer1, a linker protein that binds to the C-terminus of SHANK1. These variants may disrupt protein-protein networks in dendritic spines. Dispersed localization of the truncated SHANK1 variants within the spine and dendritic shaft was also observed when expressed in neurons, indicating impaired synaptic localization of truncated SHANK1. CONCLUSION: This report expands the clinical spectrum of individuals with truncating SHANK1 variants and describes the impact these variants may have on the pathophysiology of neurodevelopmental disorders.

Bi-allelic loss of ERGIC1 causes relatively mild arthrogryposis.

Arthrogryposis describes the presence of multiple joint-contractures. Clinical severity of this phenotype is variable, and more than 400 causative genes have been proposed. Among these, ERGIC1 is a recently reported candidate encoding a putative transmembrane protein of the ER-Golgi interface. Two homozygous missense variants have been reported in patients with relatively mild non-syndromic arthrogryposis. In a consanguineous family with two affected siblings presenting congenital arthrogryposis and some facial dysmorphism we performed prenatal array-CGH, postnatal targeted exome and genome sequencing. Genome sequencing identified a homozygous 22.6 Kb deletion encompassing the promoter and first exon of ERGIC1. mRNA quantification showed the complete absence of ERGIC1 expression in the two affected siblings and a decrease in heterozygous parents. Our observations validate the pathogenic role of ERGIC1 in congenital arthrogryposis and demonstrate that complete loss of function causes a relatively mild phenotype. These findings will contribute to improve genetic counseling of ERGIC1 mutations.

Molecular characterization of pathogenic OTOA gene conversions in hearing loss patients.

Bi-allelic loss-of-function variants of OTOA are a well-known cause of moderate-to-severe hearing loss. Whereas non-allelic homologous recombination-mediated deletions of the gene are well known, gene conversions to pseudogene OTOAP1 have been reported in the literature but never fully described nor their pathogenicity assessed. Here, we report two unrelated patients with moderate hearing-loss, who were compound heterozygotes for a converted allele and a deletion of OTOA. The conversions were initially detected through sequencing depths anomalies at the OTOA locus after exome sequencing, then confirmed with long range polymerase chain reactions. Both conversions lead to loss-of-function by introducing a premature stop codon in exon 22 (p.Glu787*). Using genomic alignments and long read nanopore sequencing, we found that the two probands carry stretches of converted DNA of widely different lengths (at least 9 kbp and around 900 bp, respectively).

Complex movement disorder in a patient with heterozygous YY1 mutation (Gabriele-de Vries syndrome).

YY1 mutations cause Gabriele-de Vries syndrome, a recently described condition involving cognitive impairment, facial dysmorphism and intrauterine growth restriction. Movement disorders were reported in 5/10 cases of the original series, but no detailed description was provided. Here we present a 21-year-old woman with a mild intellectual deficit, facial dysmorphism and a complex movement disorder including an action tremor, cerebellar ataxia, dystonia, and partial ocular apraxia as the presenting and most striking feature. Whole-exome sequencing revealed a novel heterozygous de novo mutation in YY1 [NM: 003403.4 (YY1): c.907 T > C; p.(Cys303Arg)], classified as pathogenic according to the ACMG guidelines.

LARS2-Perrault syndrome: a new case report and literature review.

BACKGROUND: Perrault syndrome is a rare recessive and genetically heterogeneous disorder characterized by sensorineural hearing loss in males and females and gonadal dysgenesis in females. Mutations in seven different genes have been identified: HARS2, HSD17B4, CLLP, C10orf, ERAL1, TWNK and LARS2. To date, 19 variants have been reported in 18 individuals with LARS2-Perrault syndrome. CASE PRESENTATION: Here we describe the case of an 8-year-old girl with compound heterozygous missense mutations in the LARS2 gene. We identified two missense mutations [c.457A > C, p.(Asn153His) and c.1565C > A, p.(Thr522Asn)] and subsequent familial segregation showed that each parent had transmitted a mutation. CONCLUSIONS: These results have implications for genetic counseling and provide insight into the functional role of LARS2. This case highlights the importance of an early diagnosis. Systematic genetic screening of children with hearing loss allows the early identification of a Perrault syndrome in order to ensure specific endocrinological surveillance and management to prevent secondary complications. Clinical data are compared with the other cases reported in the literature.

SCN8A heterozygous variants are associated with anoxic-epileptic seizures.

Anoxic-epileptic seizures (AES) are rare outcomes of common childhood reflex anoxic syncope that trigger a true epileptic seizure. The term AES was coined by Stephenson in 1983, to differentiate these events from convulsive syncopes and the more common reflex anoxic syncopes. A genetic susceptibility for AES has been postulated; but, its molecular basis has up to now been elusive. We report here two illustrative cases and show the association of de novo SCN8A variants and AES. One of them had focal or generalized seizures and autonomic symptoms triggered by orthostatism; the second had breath-holding spells triggered by pain or exercise leading to tonic-clonic seizures; both had repeatedly normal EEGs and a family history of reflex syncope. The data of three additional AES patients further suggest, for the first time, a link between SCN8A pathogenic variants and AES. The neurodevelopment of four patients was abnormal. Four of the five SCN8A mutations observed here were previously described in patients with seizure disorders. Seizures responded particularly well to sodium channel blockers. Our observation enriches the spectrum of seizures linked with SCN8A pathogenic variants.

MCF2 is linked to a complex perisylvian syndrome and affects cortical lamination.

The combination of congenital bilateral perisylvian syndrome (CBPS) with lower motor neuron dysfunction remains unusual and suggests a potential common genetic insult affecting basic neurodevelopmental processes. Here we identify a putatively pathogenic missense mutation in the MCF2 gene in a boy with CBPS. Using in utero electroporation to genetically manipulate cortical neurons during corticogenesis, we demonstrate that the mouse Mcf2 gene controls the embryonic migration of cortical projection neurons. Strikingly, we find that the CBPS-associated MCF2 mutation impairs cortical laminar positioning, supporting the hypothesis that alterations in the process of embryonic neuronal migration can lead to rare cases of CBPS.

Single cell transcriptome in aneuploidies reveals mechanisms of gene dosage imbalance.

Aneuploidy is a major source of gene dosage imbalance due to copy number alterations (CNA), and viable human trisomies are model disorders of altered gene expression. We study gene and allele-specific expression (ASE) of 9668 single-cell fibroblasts from trisomy 21 (T21) discordant twins and from mosaic T21, T18, T13 and T8. We examine 928 single cells with deep scRNAseq. Expected and observed overexpression of trisomic genes in trisomic vs. diploid bulk RNAseq is not detectable in trisomic vs. diploid single cells. Instead, for trisomic genes with low-to-average expression, their altered gene dosage is mainly due to the higher fraction of trisomic cells simultaneously expressing these genes, in agreement with a stochastic 2-state burst-like model of transcription. These results, confirmed in a further analysis of 8740 single fibroblasts with shallow scRNAseq, suggest that the specific transcriptional profile of each gene contributes to the phenotypic variability of trisomies. We propose an improved model to understand the effects of CNA and, generally, of gene regulation on gene dosage imbalance.

Exome sequencing identifies a de novo FOXA2 variant in a patient with syndromic diabetes.

OBJECTIVE: When diabetes is associated with congenital malformations, without autoimmune antibodies, a genetic cause is suspected. Here, we aimed to identify a defective gene that led to diabetes. RESEARCH DESIGN AND METHODS: We performed an exome analysis of an index case and his healthy parents. RESULTS: The child presented with childhood-onset diabetes, congenital hypopituitarism, cardiac malformation, and anal atresia. A DNA analysis revealed a heterozygous de novo pathogenic variant in the developmental transcription factor, forkhead box A2 (FOXA2). The mutation resided in the DNA-binding domain, which is highly conserved among species. Tridimensional molecular dynamics simulation modeling predicted an altered interaction between the mutated protein and DNA. CONCLUSIONS: A defect in the FOXA2 DNA-binding domain was associated with childhood-onset diabetes and multiple congenital anomalies, which reflected the pleiotropic nature of the gene. This report extends the recently described phenotype of neonatal hypoglycemia to later-onset diabetes. We suggest to include FOXA2 analysis for neonatal hypoglycemia and to implement a long-term follow-up, particularly for the risk of diabetes.

Bi-allelic Loss-of-Function Variants in DNMBP Cause Infantile Cataracts.

Infantile and childhood-onset cataracts form a heterogeneous group of disorders; among the many genetic causes, numerous pathogenic variants in additional genes associated with autosomal-recessive infantile cataracts remain to be discovered. We identified three consanguineous families affected by bilateral infantile cataracts. Using exome sequencing, we found homozygous loss-of-function variants in DNMBP: nonsense variant c.811C>T (p.Arg271∗) in large family F385 (nine affected individuals; LOD score = 5.18 at θ = 0), frameshift deletion c.2947_2948del (p.Asp983∗) in family F372 (two affected individuals), and frameshift variant c.2852_2855del (p.Thr951Metfs∗41) in family F3 (one affected individual). The phenotypes of all affected individuals include infantile-onset cataracts. RNAi-mediated knockdown of the Drosophila ortholog still life (sif), enriched in lens-secreting cells, affects the development of these cells as well as the localization of E-cadherin, alters the distribution of septate junctions in adjacent cone cells, and leads to a ∼50% reduction in electroretinography amplitudes in young flies. DNMBP regulates the shape of tight junctions, which correspond to the septate junctions in invertebrates, as well as the assembly pattern of E-cadherin in human epithelial cells. E-cadherin has an important role in lens vesicle separation and lens epithelial cell survival in humans. We therefore conclude that DNMBP loss-of-function variants cause infantile-onset cataracts in humans.

Visual impairment and progressive phthisis bulbi caused by recessive pathogenic variant in MARK3.

Developmental eye defects often severely reduce vision. Despite extensive efforts, for a substantial fraction of these cases the molecular causes are unknown. Recessive eye disorders are frequent in consanguineous populations and such large families with multiple affected individuals provide an opportunity to identify recessive causative genes. We studied a Pakistani consanguineous family with three affected individuals with congenital vision loss and progressive eye degeneration. The family was analyzed by exome sequencing of one affected individual and genotyping of all family members. We have identified a non-synonymous homozygous variant (NM_001128918.2: c.1708C > G: p.Arg570Gly) in the MARK3 gene as the likely cause of the phenotype. Given that MARK3 is highly conserved in flies (I: 55%; S: 67%) we knocked down the MARK3 homologue, par-1, in the eye during development. This leads to a significant reduction in eye size, a severe loss of photoreceptors and loss of vision based on electroretinogram (ERG) recordings. Expression of the par-1 p.Arg792Gly mutation (equivalent to the MARK3 variant found in patients) in developing fly eyes also induces loss of eye tissue and reduces the ERG signals. The data in flies and human indicate that the MARK3 variant corresponds to a loss of function. We conclude that the identified mutation in MARK3 establishes a new gene-disease link, since it likely causes structural abnormalities during eye development and visual impairment in humans, and that the function of MARK3/par-1 is evolutionarily conserved in eye development.

Novel NEXMIF pathogenic variant in a boy with severe autistic features, intellectual disability, and epilepsy, and his mildly affected mother.

Intellectual disability (ID) and autism spectrum disorders are complex neurodevelopmental disorders occurring among all ethnic and socioeconomic groups. Pathogenic variants in the neurite extension and migration factor (NEXMIF) gene (formerly named KIAA2022) on the X chromosome are responsible for ID, autistic behavior, epilepsy, or dysmorphic features in males. Most affected females described had a milder phenotype or were asymptomatic obligate carriers. We report here for the first time mother-to-son transmission of a novel NEXMIF truncating variant without X-inactivation skewing in the blood. Truncating gene variant leads to symptomatic mother to severely affected son transmission. Our findings emphasize that NEXMIF sequencing should be strongly considered in patients with unexplained autism spectrum disorder, ID, and epilepsy, irrespective of gender. Such testing could increase our knowledge of the pathogenicity of NEXMIF variants and improve genetic counseling.

Biallelic variants in KIF14 cause intellectual disability with microcephaly.

Kinesin proteins are critical for various cellular functions such as intracellular transport and cell division, and many members of the family have been linked to monogenic disorders and cancer. We report eight individuals with intellectual disability and microcephaly from four unrelated families with parental consanguinity. In the affected individuals of each family, homozygosity for likely pathogenic variants in KIF14 were detected; two loss-of-function (p.Asn83Ilefs*3 and p.Ser1478fs), and two missense substitutions (p.Ser841Phe and p.Gly459Arg). KIF14 is a mitotic motor protein that is required for spindle localization of the mitotic citron rho-interacting kinase, CIT, also mutated in microcephaly. Our results demonstrate the involvement of KIF14 in development and reveal a wide phenotypic variability ranging from fetal lethality to moderate developmental delay and microcephaly.

Biallelic variants in LINGO1 are associated with autosomal recessive intellectual disability, microcephaly, speech and motor delay.

PURPOSE: To elucidate the novel molecular cause in two unrelated consanguineous families with autosomal recessive intellectual disability. METHODS: A combination of homozygosity mapping and exome sequencing was used to locate the plausible genetic defect in family F162, while only exome sequencing was followed in the family PKMR65. The protein 3D structure was visualized with the University of California-San Francisco Chimera software. RESULTS: All five patients from both families presented with severe intellectual disability, aggressive behavior, and speech and motor delay. Four of the five patients had microcephaly. We identified homozygous missense variants in LINGO1, p.(Arg290His) in family F162 and p.(Tyr288Cys) in family PKMR65. Both variants were predicted to be pathogenic, and segregated with the phenotype in the respective families. Molecular modeling of LINGO1 suggests that both variants interfere with the glycosylation of the protein. CONCLUSION: LINGO1 is a transmembrane receptor, predominantly found in the central nervous system. Published loss-of-function studies in mouse and zebrafish have established a crucial role of LINGO1 in normal neuronal development and central nervous system myelination by negatively regulating oligodendrocyte differentiation and neuronal survival. Taken together, our results indicate that biallelic LINGO1 missense variants cause autosomal recessive intellectual disability in humans.

SERPINI1 pathogenic variants: An emerging cause of childhood-onset progressive myoclonic epilepsy.

Progressive myoclonic epilepsies are rare neurodegenerative diseases with a wide spectrum of clinical presentations and genetic heterogeneity that render their diagnosis perplexing. Discovering new imputable genes has been an ongoing process in recent years. We present two pediatric cases of progressive myoclonic epilepsy with SERPINI1 pathogenic variants that lead to a severe presentation; we highlight the importance of including this gene, previously known as causing an adult-onset dementia-epilepsy syndrome, in the genetic work-up of childhood-onset progressive myoclonic epilepsies.