Background

The causes for thousands of individually rare recessive diseases have been discovered since the adoption of next generation sequencing (NGS). Following the molecular diagnosis in older children in a family, parents could use this information to opt for fetal genotyping in subsequent pregnancies, which could inform decisions about elective termination of pregnancy. The use of NGS diagnostic sequencing in families has not been demonstrated to yield benefit in subsequent pregnancies to reduce recurrence. Here we evaluated whether genetic diagnosis in older children in families supports reduction in recurrence of recessive neurogenetic disease.

Methods

Retrospective study involving families with a child with a recessive pediatric brain disease (rPBD) that underwent NGS-based molecular diagnosis. Prenatal molecular testing was offered to couples in which a molecular diagnosis was made, to help couples seeking to prevent recurrence. With this information, families made decisions about elective termination. Pregnancies that were carried to term were assessed for the health of child and mother, and compared with historic recurrence risk of recessive disease.

Results

Between 2010 and 2016, 1172 families presented with a child a likely rPBD, 526 families received a molecular diagnosis, 91 families returned to the clinic with 101 subsequent pregnancies, and 84 opted for fetal genotyping. Sixty tested negative for recurrence for the biallelic mutation in the fetus, and all, except for one spontaneous abortion, carried to term, and were unaffected at follow-up. Of 24 that genotyped positive for the biallelic mutation, 16 were electively terminated, and 8 were carried to term and showed features of disease similar to that of the older affected sibling(s). Among the 101 pregnancies, disease recurrence in living offspring deviated from the expected 25% to the observed 12% ([95% CI 0·04 to 0·20], p = 0·011).

Conclusions

Molecular diagnosis in an older child, coupled with prenatal fetal genotyping in subsequent pregnancies and genetic counselling, allows families to make informed decisions to reduce recessive neurogenetic disease recurrence.

Pontocerebellar hypoplasia (PCH) represents a group of recessive developmental disorders characterized by impaired growth of the pons and cerebellum, which frequently follows a degenerative course. Currently, there are 10 partially overlapping clinical subtypes and 13 genes known mutated in PCH. Here, we report biallelic TBC1D23 mutations in six individuals from four unrelated families manifesting a non-degenerative form of PCH. In addition to reduced volume of pons and cerebellum, affected individuals had microcephaly, psychomotor delay, and ataxia. In zebrafish, tbc1d23 morphants replicated the human phenotype showing hindbrain volume loss. TBC1D23 localized at the trans-Golgi and was regulated by the small GTPases Arl1 and Arl8, suggesting a role in trans-Golgi membrane trafficking. Altogether, this study provides a causative link between TBC1D23 mutations and PCH and suggests a less severe clinical course than other PCH subtypes.

This study presents 46 patients from 23 unrelated Egyptian families with ALS2-related disorders without evidence of lower motor neuron involvement. Age at onset ranged from 10 months to 2.5 years, featuring progressive upper motor neuron signs. Detailed clinical phenotypes demonstrated inter- and intrafamilial variability. We identified 16 homozygous disease-causing ALS2 variants; sorted as splice-site, missense, frameshift, nonsense and in-frame in eight, seven, four, three, and one families, respectively. Seven of these variants were novel, expanding the mutational spectrum of the ALS2 gene. As expected, clinical severity was positively correlated with disease onset (p = 0.004). This work provides clinical and molecular profiles of a large single ethnic cohort of patients with ALS2 mutations, and suggests that infantile ascending hereditary spastic paralysis (IAHSP) and juvenile primary lateral sclerosis (JPLS) are belonged to one entity with no phenotype-genotype correlation.

TMEM161B encodes an evolutionarily conserved widely expressed novel 8-pass transmembrane protein of unknown function in human. Here we identify TMEM161B homozygous hypomorphic missense variants in our recessive polymicrogyria (PMG) cohort. Patients carrying TMEM161B mutations exhibit striking neocortical PMG and intellectual disability. Tmem161b knockout mice fail to develop midline hemispheric cleavage, whereas knock-in of patient mutations and patient-derived brain organoids show defects in apical cell polarity and radial glial scaffolding. We found that TMEM161B modulates actin filopodia, functioning upstream of the Rho-GTPase CDC42. Our data link TMEM161B with human PMG, likely regulating radial glia apical polarity during neocortical development.

Aminoacyl-tRNA synthetases (ARSs) function to transfer amino acids to cognate tRNA molecules, which are required for protein translation. To date, biallelic mutations in 31 ARS genes are known to cause recessive, early-onset severe multi-organ diseases. VARS encodes the only known valine cytoplasmic-localized aminoacyl-tRNA synthetase. Here, we report seven patients from five unrelated families with five different biallelic missense variants in VARS. Subjects present with a range of global developmental delay, epileptic encephalopathy and primary or progressive microcephaly. Longitudinal assessment demonstrates progressive cortical atrophy and white matter volume loss. Variants map to the VARS tRNA binding domain and adjacent to the anticodon domain, and disrupt highly conserved residues. Patient primary cells show intact VARS protein but reduced enzymatic activity, suggesting partial loss of function. The implication of VARS in pediatric neurodegeneration broadens the spectrum of human diseases due to mutations in tRNA synthetase genes.

Abstract: LNPK encodes a conserved membrane protein that stabilizes the junctions of the tubular endoplasmic reticulum network playing crucial roles in diverse biological functions. Recently, homozygous variants in LNPK were shown to cause a neurodevelopmental disorder (OMIM#618090) in four patients displaying developmental delay, epilepsy, and non-specific brain malformations including corpus callosum hypoplasia and variable impairment of cerebellum. We sought to delineate the molecular and phenotypic spectrum of LNPK-related disorder. Exome or genome sequencing was carried out in eleven families. Thorough clinical and neuroradiological evaluation was performed for all the affected individuals, including review of previously reported patients. We identified twelve distinct homozygous loss-of-function variants in sixteen individuals presenting with moderate to profound developmental delay, cognitive impairment, regression, refractory epilepsy and a recognizable neuroimaging pattern consisting of corpus callosum hypoplasia and signal alterations of the forceps minor (“ear-of-the-lynx” sign), variably associated with substantia nigra signal alterations, mild brain atrophy, short midbrain, and cerebellar hypoplasia/atrophy. In summary, we define the core phenotype of LNPK-related disorder and expand the list of neurological disorders presenting with the “ear of the lynx” sign suggesting a possible common underlying mechanism related to endoplasmic reticulum-phagy dysfunction.

Trafficking protein particle (TRAPP) complexes, which include the TRAPPC4 protein, regulate membrane trafficking between lipid organelles in a process termed vesicular tethering. TRAPPC4 was recently implicated in a recessive neurodevelopmental condition in four unrelated families due to a shared c.454+3A>G splice variant. Here, we report 23 patients from 17 independent families with an early-infantile-onset neurodegenerative presentation, where we also identified the homozygous variant hg38:11:119020256 A>G (NM_016146.5:c.454+3A>G) in TRAPPC4 through exome or genome sequencing. No other clinically relevant TRAPPC4 variants were identified among any of over 10,000 patients with neurodevelopmental conditions. We found the carrier frequency of TRAPPC4 c.454+3A>G was 2.4-5.4 per 10,000 healthy individuals. Affected individuals with the homozygous TRAPPC4 c.454+3A>G variant showed profound psychomotor delay, developmental regression, early-onset epilepsy, microcephaly and progressive spastic tetraplegia. Based upon RNA sequencing, the variant resulted in partial exon 3 skipping and generation of an aberrant transcript owing to use of a downstream cryptic splice donor site, predicting a premature stop codon and nonsense mediated decay. These data confirm the pathogenicity of the TRAPPC4 c.454+3A>G variant, and refine the clinical presentation of TRAPPC4-related encephalopathy.

Autosomal-recessive cerebellar hypoplasia and ataxia constitute a group of heterogeneous brain disorders caused by disruption of several fundamental cellular processes. Here, we identified 10 families showing a neurodegenerative condition involving pontocerebellar hypoplasia with microcephaly (PCHM). Patients harbored biallelic mutations in genes encoding the spliceosome components Peptidyl-Prolyl Isomerase Like-1 (PPIL1) or Pre-RNA Processing-17 (PRP17). Mouse knockouts of either gene were lethal in early embryogenesis, whereas PPIL1 patient mutation knockin mice showed neuron-specific apoptosis. Loss of either protein affected splicing integrity, predominantly affecting short and high GC-content introns and genes involved in brain disorders. PPIL1 and PRP17 form an active isomerase-substrate interaction, but we found that isomerase activity is not critical for function. Thus, we establish disrupted splicing integrity and "major spliceosome-opathies" as a new mechanism underlying PCHM and neurodegeneration and uncover a non-enzymatic function of a spliceosomal proline isomerase.