Two novel cases of biallelic SMPD4 variants with brain structural abnormalities.

Sphingomyelin phosphodiesterase 4 (SMPD4) encodes a member of the Mg2+-dependent, neutral sphingomyelinase family that catalyzes the hydrolysis of the phosphodiester bond of sphingomyelin to form phosphorylcholine and ceramide. Recent studies have revealed that biallelic loss-of-function variants of SMPD4 cause syndromic neurodevelopmental disorders characterized by microcephaly, congenital arthrogryposis, and structural brain anomalies. In this study, three novel loss-of-function SMPD4 variants were identified using exome sequencing (ES) in two independent patients with developmental delays, microcephaly, seizures, and brain structural abnormalities. Patient 1 had a homozygous c.740_741del, p.(Val247Glufs*21) variant and showed profound intellectual disability, hepatomegaly, a simplified gyral pattern, and a thin corpus callosum without congenital dysmorphic features. Patient 2 had a compound heterozygous nonsense c.2124_2125del, p.(Phe709*) variant and splice site c.1188+2dup variant. RNA analysis revealed that the c.1188+2dup variant caused exon 13 skipping, leading to a frameshift (p.Ala406Ser*6). In vitro transcription analysis using minigene system suggested that mRNA transcribed from mutant allele may be degraded by nonsense-mediated mRNA decay system. He exhibited diverse manifestations, including growth defects, muscle hypotonia, respiratory distress, arthrogryposis, insulin-dependent diabetes mellitus, sensorineural hearing loss, facial dysmorphism, and various brain abnormalities, including cerebral atrophy, hypomyelination, and cerebellar hypoplasia. Here, we review previous literatures and discuss the phenotypic diversity of SMPD4-related disorders.

SMPD4 regulates mitotic nuclear envelope dynamics and its loss causes microcephaly and diabetes.

Biallelic loss-of-function variants in SMPD4 cause a rare and severe neurodevelopmental disorder with progressive congenital microcephaly and early death. SMPD4 encodes a sphingomyelinase that hydrolyses sphingomyelin into ceramide at neutral pH and can thereby affect membrane lipid homeostasis. SMPD4 localizes to the membranes of the endoplasmic reticulum and nuclear envelope and interacts with nuclear pore complexes (NPC). We refine the clinical phenotype of loss-of-function SMPD4 variants by describing five individuals from three unrelated families with longitudinal data due to prolonged survival. All individuals surviving beyond infancy developed insulin-dependent diabetes, besides presenting with a severe neurodevelopmental disorder and microcephaly, making diabetes one of the most frequent age-dependent non-cerebral abnormalities. We studied the function of SMPD4 at the cellular and organ levels. Knock-down of SMPD4 in human neural stem cells causes reduced proliferation rates and prolonged mitosis. Moreover, SMPD4 depletion results in abnormal nuclear envelope breakdown and reassembly during mitosis and decreased post-mitotic NPC insertion. Fibroblasts from affected individuals show deficient SMPD4-specific neutral sphingomyelinase activity, without changing (sub)cellular lipidome fractions, which suggests a local function of SMPD4 on the nuclear envelope. In embryonic mouse brain, knockdown of Smpd4 impairs cortical progenitor proliferation and induces premature differentiation by altering the balance between neurogenic and proliferative progenitor cell divisions. We hypothesize that, in individuals with SMPD4-related disease, nuclear envelope bending, which is needed to insert NPCs in the nuclear envelope, is impaired in the absence of SMPD4 and interferes with cerebral corticogenesis and survival of pancreatic beta cells.

Loss of SMPD4 Causes a Developmental Disorder Characterized by Microcephaly and Congenital Arthrogryposis.

Sphingomyelinases generate ceramide from sphingomyelin as a second messenger in intracellular signaling pathways involved in cell proliferation, differentiation, or apoptosis. Children from 12 unrelated families presented with microcephaly, simplified gyral pattern of the cortex, hypomyelination, cerebellar hypoplasia, congenital arthrogryposis, and early fetal/postnatal demise. Genomic analysis revealed bi-allelic loss-of-function variants in SMPD4, coding for the neutral sphingomyelinase-3 (nSMase-3/SMPD4). Overexpression of human Myc-tagged SMPD4 showed localization both to the outer nuclear envelope and the ER and additionally revealed interactions with several nuclear pore complex proteins by proteomics analysis. Fibroblasts from affected individuals showed ER cisternae abnormalities, suspected for increased autophagy, and were more susceptible to apoptosis under stress conditions, while treatment with siSMPD4 caused delayed cell cycle progression. Our data show that SMPD4 links homeostasis of membrane sphingolipids to cell fate by regulating the cross-talk between the ER and the outer nuclear envelope, while its loss reveals a pathogenic mechanism in microcephaly.

A patient with compound heterozygosity of SMPD4: Another example of utility of exome-based copy number analysis in autosomal recessive disorders.

For the efficient diagnosis of rare and undiagnosed diseases, the parallel detection of copy number variants (CNVs) and single nucleotide variants using exome analysis is required. Recently, our group reported the usefulness of a program called EXCAVATOR2, which screens for CNVs from aligned exome data in bam format. This method is expected to contribute to the identification of structural variants and to improve the diagnosis rate, especially for the diagnosis of autosomal recessive disease, when a conventional exome analysis identifies a pathogenic variant in one allele but not the other. Here we report a 2-year-old Japanese boy with an undiagnosed disease. He had severe neonatal asphyxia, severe intellectual disability, intractable seizures, cerebellar and brainstem hypoplasia and dysmorphic features including a prominent supraorbital ridge, thin upper lip, and prominent antihelix. An exome analysis reinforced with a copy number analysis using the EXCAVATOR2 method revealed that the patient had a hemizygous variant in chr2(GRCh37):g.130925108G>A, NM_017951.4 c.832C>T, p.(Arg278*) in SMPD4 that was derived from his father and a deletion of SMPD4 derived from his mother. The presence of the deletion spanning SMPD4 was confirmed by short-read and long-read whole-genome sequencing. The successful diagnosis of this reported patient demonstrates the diagnostic utility of EXCAVATOR2 and overcomes the weakness of exome analysis for the detection of autosomal recessive diseases in nonconsanguineous families, significantly impacting genetic counseling for family planning.

Generating Membrane Curvature at the Nuclear Pore: A Lipid Point of View.

In addition to its structural role in enclosing and protecting the genome, the nuclear envelope (NE) forms a highly adaptive communication interface between the cytoplasm and the nuclear interior in eukaryotic cells. The double membrane of the NE is perforated by nuclear pores lined with large multi-protein structures, called nuclear-pore complexes (NPCs), which selectively allow the bi-directional transport of ions and macromolecular cargo. In order to nucleate a pore, the inner and outer nuclear membrane have to fuse at the site of NPC insertion, a process requiring both lipid bilayers to be deformed into highly curved structures. How this curvature is achieved and which factors are involved in inducing and stabilizing membrane curvature at the nuclear pore remain largely unclear. In this review, we will summarize the molecular mechanisms thought to be involved in membrane curvature generation, with a particular emphasis on the role of lipids and lipid metabolism in shaping the nuclear pore membrane.

Proximity Ligation Mapping of Microcephaly Associated SMPD4 Shows Association with Components of the Nuclear Pore Membrane.

SMPD4 is a neutral sphingomyelinase implicated in a specific type of congenital microcephaly. Although not intensively studied, SMPD4 deficiency has also been found to cause cell division defects. This suggests a role for SMPD4 in cell-cycle and differentiation. In order to explore this role, we used proximity ligation to identify the partners of SMPD4 in vivo in HEK293T cells. We found that these partners localize near the endoplasmic reticulum (ER) and the nuclear membrane. Using mass spectrometry, we could identify these partners and discovered that SMPD4 is closely associated with several nucleoporins, including NUP35, a nucleoporin directly involved in pore membrane curvature and pore insertion. This suggests that SMPD4 may play a role in this process.

Case Report: Novel Biallelic Null Variants of SMPD4 Confirm Its Involvement in Neurodevelopmental Disorder With Microcephaly, Arthrogryposis, and Structural Brain Anomalies.

The SMPD4 gene encodes sphingomyelin phosphodiesterase 4, which preferentially hydrolyzes sphingomyelin over other phospholipids. The biallelic loss-of-function variants of SMPD4 have been identified in a group of children with neurodevelopmental disorder with microcephaly, arthrogryposis, and structural brain anomalies (NEDMABA). Here, we report a girl of Chinese ancestry with intrauterine growth restriction, microcephaly, postnatal developmental delay, arthrogryposis, hypertonicity, seizure, and hypomyelination on brain magnetic resonance imaging; biallelic null variants (c.1347C > G [p.Tyr449*]; Chr2 [GRCh37]: g.130877574_131221737del [whole-gene deletion]) were detected by whole-exome sequencing. Our case is the first report of NEDMABA of Chinese ancestry, confirming the involvement of SMPD4 in NEDMABA and expanding the mutation spectrum of this syndrome.

Multidisciplinary interaction and MCD gene discovery. The perspective of the clinical geneticist.

The increasing pace of gene discovery in the last decade has brought a major change in the way the genetic causes of brain malformations are being diagnosed. Unbiased genomic screening has gained the first place in the diagnostic protocol of a child with congenital (brain) anomalies and the detected variants are matched with the phenotypic presentation afterwards. This process is defined as "reverse phenotyping". Screening of DNA, through copy number variant analysis of microarrays and analysis of exome data on different platforms, obtained from the index patient and both parents has become a routine approach in many centers worldwide. Clinicians are used to multidisciplinary team interaction in patient care and disease management and this explains why the majority of research that has led to the discovery of new genetic disorders nowadays proceeds from clinical observations to genomic analysis and to data exchange facilitated by open access sharing databases. However, the relevance of multidisciplinary team interaction has not been object of systematic research in the field of brain malformations. This review will illustrate some examples of how diagnostically driven questions through multidisciplinary interaction, among clinical and preclinical disciplines, can be successful in the discovery of new genes related to brain malformations. The first example illustrates the setting of interaction among neurologists, geneticists and neuro-radiologists. The second illustrates the importance of interaction among clinical dysmorphologists for pattern recognition of syndromes with multiple congenital anomalies. The third example shows how fruitful it can be to step out of the "clinical comfort zone", and interact with basic scientists in applying emerging technologies to solve the diagnostic puzzles.