Exploring unsolved cases of lissencephaly spectrum: integrating exome and genome sequencing for higher diagnostic yield.

Lissencephaly is a rare brain malformation characterized by abnormal neuronal migration during cortical development. In this study, we performed a comprehensive genetic analysis using next-generation sequencing in 12 unsolved Japanese lissencephaly patients, in whom PAFAH1B1, DCX, TUBA1A, and ARX variants were excluded using the Sanger method. Exome sequencing (ES) was conducted on these 12 patients, identifying pathogenic variants in CEP85L, DYNC1H1, LAMC3, and DCX in four patients. Next, we performed genome sequencing (GS) on eight unsolved patients, and structural variants in PAFAH1B1, including an inversion and microdeletions involving several exons, were detected in three patients. Notably, these microdeletions in PAFAH1B1 could not to be detected by copy number variation (CNV) detection tools based on the depth of coverage methods using ES data. The density of repeat sequences, including Alu sequences or segmental duplications, which increase the susceptibility to structural variations, is very high in some lissencephaly spectrum genes (PAFAH1B1, TUBA1A, DYNC1H1). These missing CNVs were due to the limitations of detecting repeat sequences in ES-based CNV detection tools. Our study suggests that a combined approach integrating ES with GS can contribute to a higher diagnostic yield and a better understanding of the genetic landscape of the lissencephaly spectrum.

A family with neuronal intranuclear inclusion disease with focal segmental glomerulosclerosis.

BACKGROUND: Neuronal intranuclear inclusion disease (NIID) is a rare neurodegenerative disease caused by the expansion of GGC repeats in the 5'-untranslated region (5'-UTR) of NOTCH2NLC. Although increasing evidence suggests that NIID affects various organs, its association with renal involvement remains unclear. We studied the genetic background of a family with NIID, in which four of five members presented with proteinuria as the initial manifestation. The renal pathology of three patients was diagnosed as focal segmental glomerulosclerosis (FSGS) at a previous hospital. These patients also presented with tremors, retinal degeneration, and episodic neurological events. Finally, one patient exhibited reversible bilateral thalamic high-intensity signal changes on diffusion-weighted imaging during episodic neurological events. METHODS: Exome sequencing (ES) and nanopore long-read whole-genome sequencing (LR-WGS) were performed on the index case, followed by nanopore target sequencing using Cas9-mediated PCR-free enrichment and methylation analysis. RESULTS: ES revealed no candidate variants; however, nanopore LR-WGS in the index case revealed expansion of short tandem repeats (STR) in NOTCH2NLC. Subsequent nanopore target sequencing using Cas9-mediated PCR-free enrichment showed STR expansion of NOTCH2NLC in an affected sibling and asymptomatic father. Methylation analysis using nanopore data revealed hypermethylation of the expanded allele in the asymptomatic father and partial hypermethylation in a mildly symptomatic sibling, whereas the expanded allele was hypomethylated in the index case. CONCLUSIONS: This investigation expands the clinical spectrum of NIID, suggesting that STR expansion of NOTCH2NLC is a cause of renal diseases, including FSGS.

A novel heterozygous TMEM63A variant in a familial case with early onset nystagmus, severe hypomyelination, and a favorable adult prognosis.

Heterozygous transmembrane protein 63A (TMEM63A) variants cause transient infantile hypomyelinating leukodystrophy-19, which features remarkable natural resolution of clinical and imaging findings during childhood. Previous reports have mainly described de novo variants lacking detailed familial cases. Herein, we describe the clinical course of familial cases with a TMEM63A variant. A 5-month-old girl presented with nystagmus, global hypotonia, and difficulty swallowing since birth. Brain magnetic resonance imaging at 1.5 and 5 months revealed diffuse hypomyelination. Her mother, maternal aunt, and grandfather had nystagmus and motor developmental delays in infancy, which resolved spontaneously during childhood. Compared with these cases, the proband's motor developmental delay was profound, and she was the only one with feeding difficulties, necessitating nasogastric tube feeding. Genetic testing revealed a heterozygous TMEM63A variant (NM_014698.3:c.1658G>A, p.(Gly553Asp)) in the proband and her family. This is the first three-generation familial report of a TMEM63A variant that provides insight into its history and heterogeneity.

Chloride Voltage-Gated Channel 2 (CLCN2)-Related Leukoencephalopathy Exhibiting Reduced Choline Levels on Magnetic Resonance Spectroscopy.

In this article, we report the third case of chloride voltage-gated channel 2 (CLCN2)-related leukoencephalopathy (CC2L) in Japan. The patient presented with headache, vertigo, and mild visual impairment. The CLCN2 variant of the patient, NM_004366.6:c.61dup, p.(Leu21Profs*27), was also found in two other Japanese patients as this variant is relatively common in the Japanese population. Magnetic resonance imaging (MRI) revealed T2 prolongation with reduced diffusion in the bilateral posterior limbs of the internal capsule, cerebral peduncles, and superior and middle cerebellar peduncles. Magnetic resonance spectroscopy (MRS) of normal-appearing white matter revealed decreased choline content. This represents the first evidence of decreased choline levels in CC2L, highlighting the superior sensitivity of MRS over MRI.

Spontaneous Remission of Epileptic Seizures Following Norovirus Infection in a Patient With DNM1 Encephalopathy.

Epileptic seizures can be worsened by infections; however, they sometimes disappear or decrease after an acute viral infection, although this is rare. We report the spontaneous remission of epileptic seizures following norovirus-induced viral gastroenteritis in a boy with DNM1 encephalopathy. He had clonic seizures daily from the age of two months and developed epileptic spasms at 14 months of age; he was admitted to the hospital at this time. A physical examination revealed hypotonia, strabismus, tongue protrusion with drooping, and widely spaced teeth. Although brain magnetic resonance imaging was unremarkable, electroencephalography revealed frequent occipital spikes. Three days after admission, the patient developed frequent diarrhea without a fever. A rapid immunochromatographic test of norovirus in a stool sample was positive. Immediately after the appearance of diarrhea, the epileptic seizures disappeared. Currently, at the age of five years, the patient has a profound psychomotor developmental delay; he has no verbal expression and is unable to walk. He has experienced involuntary movements of the myoclonus since 10 months of age. Whole-exome sequencing of the patient's DNA revealed the presence of a heterozygous de novo variant of DNM1: c.709C>T (p.Arg237Trp). Although the findings from our patient suggest that underlying neural network abnormalities were ameliorated by immunological mechanisms as a result of the viral infection, further research is needed to clarify the mechanisms behind this spontaneous remission of seizures.

Pathogenic sequence variant and microdeletion affecting HMGA2 in Silver-Russell syndrome: case reports and literature review.

Silver-Russell syndrome (SRS) is a representative imprinting disorder characterized by pre- and postnatal growth failure. We encountered two Japanese SRS cases with a de novo pathogenic frameshift variant of HMGA2 (NM_003483.6:c.138_141delinsCT, p.(Lys46Asnfs*16)) and a de novo ~ 3.4 Mb microdeletion at 12q14.2-q15 involving HMGA2, respectively. Furthermore, we compared clinical features in previously reported patients with various genetic conditions leading to compromised IGF2 expression, i.e., HMGA2 aberrations, PLAG1 aberrations, IGF2 aberrations, and H19/IGF2:IG-DMR epimutations (hypomethylations). The results provide further support for HMGA2 being involved in the development of SRS and imply some characteristic features in patients with HMGA2 aberrations.

Long-term clinical observation of patients with heterozygous KIF1A variants.

KIF1A-related disorders (KRDs) encompass recessive and dominant variants with wide clinical variability. Recent genetic investigations have expanded the clinical phenotypes of heterozygous KIF1A variants. However, there have been a few long-term observational studies of patients with heterozygous KIF1A variants. A retrospective chart review of consecutive patients diagnosed with spastic paraplegia at Miyagi Children's Hospital from 2016 to 2020 identified six patients with heterozygous KIF1A variants. To understand the long-term changes in clinical symptoms, we examined these patients in terms of their characteristics, clinical symptoms, results of electrophysiological and neuroimaging studies, and genetic testing. The median follow-up period was 30 years (4-44 years). This long-term observational study showed that early developmental delay and equinus gait, or unsteady gait, are the first signs of disease onset, appearing with the commencement of independent walking. In addition, later age-related progression was observed in spastic paraplegia, and the appearance of axonal neuropathy and reduced visual acuity were characteristic features of the late disease phenotype. Brain imaging showed age-related progression of cerebellar atrophy and the appearance of hyperintensity of optic radiation on T2WI and FLAIR imaging. Long-term follow-up revealed a pattern of steady progression and a variety of clinical symptoms, including spastic paraplegia, peripheral neuropathy, reduced visual acuity, and some degree of cerebellar ataxia. Clinical variability between patients was observed to some extent, and therefore, further studies are required to determine the phenotype-genotype correlation.

Case Report: Novel compound heterozygous TPRKB variants cause Galloway-Mowat syndrome.

Background: Galloway-Mowat syndrome (GAMOS) is a rare genetic disease characterized by early-onset nephrotic syndrome and microcephaly with central nervous system abnormalities. Pathogenic variants in genes encoding kinase, endopeptidase, and other proteins of small size (KEOPS) complex subunits cause GAMOS. The subunit TPRKB (TP53RK binding protein) has been reported in only two patients with GAMOS with homozygous missense variants. Clinical report: Herein, we described a three-year-old male with GAMOS. He exhibited developmental delay, developmental regression, microcephaly, distinctive facial features, skeletal abnormalities, and epilepsy. Brain magnetic resonance imaging revealed progressive brain atrophy, delayed myelination, T2-hypointense signals in the thalamus, and multiple intracranial abnormal signals on diffusion-weighted imaging. He presented with relapsing nephrotic proteinuria exacerbated by upper respiratory tract infections and progressive renal function decline. Exome sequencing identified compound heterozygous missense and frameshift variants in TPRKB: c.224dup, p.(Ser76IlefsTer3) and c.247C>T, p.(Leu83Phe). Conclusions: Our study supports that pathogenic TPRKB variants cause KEOPS complex-related GAMOS.

Cerebellar Ataxia With Neuropathy and Vestibular Areflexia Syndrome Due to Replication Factor C Subunit 1 Gene Repeat Expansion.

ABSTRACT: A 56-year-old man was born to consanguineous parents. He experienced slow-progressing sensory disturbances in the upper extremities. T1-weighted images showed cerebellar atrophy. 123I-IMP SPECT revealed reduced cerebral blood flow in the cerebellum. 123I-FP-CIT SPECT showed low uptake of dopamine transporter in the bilateral tail of the striatum. 123I-MIBG scintigraphy shows a decreased heart-to-mediastinum ratio. Flanking polymerase chain reaction suggested biallelic repeat expansion in intron 2 of RFC1, and subsequent repeat-primed polymerase chain reaction revealed ACAGG repeat expansion. Thus, he was diagnosed as cerebellar ataxia with neuropathy and vestibular areflexia syndrome.

Novel compound heterozygous ATP1A2 variants in a patient with fetal akinesia/hypokinesia sequence.

ATP1A2 encodes a subunit of sodium/potassium-transporting adenosine triphosphatase (Na+ /K+ -ATPase). Heterozygous pathogenic variants of ATP1A2 cause familial hemiplegic migraine, alternating hemiplegia of childhood, and developmental and epileptic encephalopathy. Biallelic loss-of-function variants in ATP1A2 lead to fetal akinesia, respiratory insufficiency, microcephaly, polymicrogyria, and dysmorphic facies, resulting in fetal death. Here, we describe a patient with compound heterozygous ATP1A2 variants consisting of missense and nonsense variants. He survived after birth with brain malformations and the fetal akinesia/hypokinesia sequence. We report a novel type of compound heterozygous variant that might extend the disease spectrum of ATP1A2.

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.

Splicing variant of WDR37 in a case of Neurooculocardiogenitourinary syndrome.

BACKGROUND: Neurooculocardiogenitourinary syndrome (NOCGUS), a multisystemic syndrome characterized by motor disorder, intellectual disability, seizures, abnormal brain structure, ocular diseases, and cardiac diseases, has been reported with missense variant of WD repeat-containing protein 37 (WDR37) in humans. This report aimed to identify the cause of NOCGUS in an affected patient. CASE PRESENTATION: We identified a de novo intronic 4-bp deletion of WDR37, c.727-27_727-24del, which were predicted to cause abnormal splicing by SpliceAI, in the patient with NOCGUS. Reverse transcription polymerase chain reaction (RT-PCR) revealed intron retention of 63 base pairs before exon 10 in messenger RNA, which was predicted to insert 21 additional aberrant amino acids (p.S242_I243insLCQKKLKISRKCLFWPSLWQQ). The patient had novel phenotypes, anal atresia, and polycystic kidney, in addition to intellectual disability, seizures, cerebellar vermian anomaly, and coloboma, which are typical in NOCGUS. We did not observe motor impairments or cardiovascular anomalies. CONCLUSION: This is the first reported case of NOCGUS with the splicing variant of WDR37, which manifests with distinctive but variable features. Our findings may expand a possible phenotypic expression of NOCGUS.

TBX5 pathogenic variant in a patient with congenital heart defect and tracheal stenosis.

Congenital tracheal stenosis is a rare life-threatening disorder caused by narrow O-shaped tracheal ring without smooth muscle. Its underlying genetic cause has not been elucidated. We performed whole exome sequencing in a patient with congenital tracheal stenosis and congenital heart defect, and identified a de novo pathogenic TBX5 variant (NM_181486.4:c.680T>C, p.(Ile227Thr)). The Ile227Thr-TBX5 protein was predicted to have a decreased stability by in silico protein structural analyses, and was shown to have a significantly reduced activity for the NPPA promoter by luciferase assay. The results, together with the expression of mouse Tbx5 in the lung and trachea and the development of tracheal cartilage dysplasia in the lung-specific Tbx5 null mice, imply the relevance of TBX5 pathogenic variants to congenital tracheal stenosis.

Sotos syndrome with marked overgrowth in three Japanese patients with heterozygous likely pathogenic NSD1 variants: case reports with review of literature.

We report three Japanese patients with Sotos syndrome accompanied by marked overgrowth, i.e., a 2 8/12-year-old boy with a height of 105.2 cm (+4.4 SD) (patient 1), the mother of patient 1 with a height of 180.8 cm (+4.1 SD) (patient 2), and a 12 10/12-year-old girl with a height of 189.4 cm (+6.3 SD) (patient 3). In addition to the marked overgrowth (tall stature), patients 1-3 exhibited Sotos syndrome-compatible macrocephaly and characteristic features, whereas intellectual and developmental disabilities remained at a borderline level in patient 1 and were apparently absent from patients 2 and 3. Thus, whole exome sequencing was performed to confirm the diagnosis, revealing a likely pathogenic c.6356A>G:p.(Asp2119Gly) variant in NSD1 of patients 1 and 2, and a likely pathogenic c.6599dupT:p.(Ser2201Valfs*4) variant in NSD1 of patient 3 (NM_022455.5). The results, in conjunction with the previously reported data in nine patients with marked overgrowth (≥4.0 SD), imply that several patients with Sotos syndrome have extreme tall stature even in adulthood. Thus, it is recommended to examine NSD1 in patients with marked overgrowth as the salient feature.

RNA sequencing and target long-read sequencing reveal an intronic transposon insertion causing aberrant splicing.

More than half of cases with suspected genetic disorders remain unsolved by genetic analysis using short-read sequencing such as exome sequencing (ES) and genome sequencing (GS). RNA sequencing (RNA-seq) and long-read sequencing (LRS) are useful for interpretation of candidate variants and detection of structural variants containing repeat sequences, respectively. Recently, adaptive sampling on nanopore sequencers enables target LRS more easily. Here, we present a Japanese girl with premature chromatid separation (PCS)/mosaic variegated aneuploidy (MVA) syndrome. ES detected a known pathogenic maternal heterozygous variant (c.1402-5A>G) in intron 10 of BUB1B (NM_001211.6), a known responsive gene for PCS/MVA syndrome with autosomal recessive inheritance. Minigene splicing assay revealed that almost all transcripts from the c.1402-5G allele have mis-splicing with 4-bp insertion. GS could not detect another pathogenic variant, while RNA-seq revealed abnormal reads in intron 2. To extensively explore variants in intron 2, we performed adaptive sampling and identified a paternal 3.0 kb insertion. Consensus sequence of 16 reads spanning the insertion showed that the insertion consists of Alu and SVA elements. Realignment of RNA-seq reads to the new reference sequence containing the insertion revealed that 16 reads have 5' splice site within the insertion and 3' splice site at exon 3, demonstrating causal relationship between the insertion and aberrant splicing. In addition, immunoblotting showed severely diminished BUB1B protein level in patient derived cells. These data suggest that detection of transcriptomic abnormalities by RNA-seq can be a clue for identifying pathogenic variants, and determination of insert sequences is one of merits of LRS.

Diagnostic MR imaging features of hypomyelination of early myelinating structures: A case report.

Hypomyelination of early myelinating structures (HEMS) has recently been defined as a new genetic disorder accompanied by clinical and MR imaging characteristics. However, no studies have focused on diffusion-weighted imaging (DWI) findings of HEMS. We would like to propose a "sheep sign," which is formed by DWI hyperintensity in the medial medullary lamina along with alternating high-low-high (HLH) intensity stripes in the posterior limb of the internal capsule. We believe the presence of the "sheep sign" on DWI in combination with alternating HLH intensity stripes may be a valuable tool for diagnosing HEMS.

A case of infantile spasms with three possibly pathogenic de novo missense variants in NF1 and GABBR1.

Neurofibromatosis type 1 (NF1) is one of the most common hereditary neurocutaneous disorders. Here, we report a unique case of a patient with typical NF1 findings and infantile spasms who had three possibly pathogenic de novo variants, c.3586C>T, p.(Leu1196Phe) and c.3590C>T, p.(Ala1197Val) in NF1 located in cis and c.1042G>C, p.(Ala348Pro) in GABBR1. This study contributes to our understanding of the effect of two cis variants on NF1 phenotypes and GABBR1-related neuropsychiatric disorders.

The Structural Abnormalities Are Deeply Involved in the Cause of RPGRIP1-Related Retinal Dystrophy in Japanese Patients.

Leber congenital amaurosis (LCA) is the most severe form of inherited retinal dystrophy. RPGRIP1-related LCA accounts for 5-6% of LCA. We performed whole-exome sequencing and whole-genome sequencing (WGS) on 29 patients with clinically suspected LCA and examined ophthalmic findings in patients with biallelic pathogenic variants of RPGRIP1. In addition to five previously reported cases, we identified five cases from four families with compound heterozygous RPGRIP1 variants using WGS. Five patients had null variants comprising frameshift variants, an Alu insertion, and microdeletions. A previously reported 1339 bp deletion involving exon 18 was found in four cases, and the deletion was relatively prevalent in the Japanese population (allele frequency: 0.002). Microdeletions involving exon 1 were detected in four cases. In patients with RPGRIP1 variants, visual acuity remained low, ranging from light perception to 0.2, and showed no correlation with age. In optical coherence tomography images, the ellipsoid zone (EZ) length decreased with age in all but one case of unimpaired EZ. The retinal structure was relatively preserved in all cases; however, there were cases with great differences in visual function compared to their siblings and a 56-year-old patient who still had a faint EZ line. Structural abnormalities may be important genetic causes of RPGRIP1-related retinal dystrophy in Japanese patients, and WGS was useful for detecting them.

Serum steroid metabolite profiling by LC-MS/MS in two phenotypic male patients with HSD17B3 deficiency: Implications for hormonal diagnosis.

Although 17ß-hydroxysteroid dehydrogenase type 3 (HSD17B3) deficiency is diagnosed when a testosterone/androstenedione (T/A-dione) ratio after human chorionic gonadotropin (hCG) stimulation is below 0.8, this cut-off value is primarily based on hormonal data measured by conventional immunoassay (IA) in patients with feminized or ambiguous genitalia. We examined two 46,XY Japanese patients with undermasculinized genitalia including hypospadias (patient 1 and patient 2). Endocrine studies by IA showed well increased serum T value after hCG stimulation (2.91 ng/mL) and a high T/A-dione ratio (4.04) in patient 1 at 2 weeks of age and sufficiently elevated basal serum T value (2.60 ng/mL) in patient 2 at 1.5 months of age. Despite such partial androgen insensitivity syndrome-like findings, whole exome sequencing identified biallelic ″pathogenic″ or ″likely pathogenic″ variants in HSD17B3 (c .188 C>T:p.(Ala63Val) and c .194 C>T:p.(Ser65Leu) in patient 1, and c.139 A>G:p.(Met47Val) and c.672 + 1 G>A in patient 2) (NM_000197.2), and functional analysis revealed reduced HSD17B3 activities of the missense variants (∼ 43% for p.Met47Val, ∼ 14% for p.Ala63Val, and ∼ 0% for p.Ser65Leu). Thus, we investigated hCG-stimulated serum steroid metabolite profiles by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in patient 1 at 7 months of age and in patient 2 at 11 months of age as well as in five control males with idiopathic micropenis aged 1 - 8 years, and found markedly high T/A-dione ratios (12.3 in patient 1 and 5.4 in patient 2) which were, however, obviously lower than those in the control boys (25.3 - 56.1) and sufficiently increased T values comparable to those of control males. The elevated T/A-dione ratios are considered be due to the residual HSD17B3 function and the measurement by LC-MS/MS. Thus, it is recommended to establish the cut-off value for the T/A-dione ratio according to the phenotypic sex reflecting the residual function and the measurement method.

Case report: Progressive pulmonary artery hypertension in a case of megalencephaly-capillary malformation syndrome.

Megalencephaly-capillary malformation syndrome (MCAP, OMIM # 602501) is caused by hyperactivity of the thephosphoinositide-3-kinase (PI3K)-Vakt murine thymoma viral oncogene homolog (AKT)-mammalian target of rapamycin (mTOR) pathway, which results in megalencephaly, capillary malformations, asymmetrical overgrowth, and connective tissue dysplasia. Herein, we report the case of a 7-month-old girl with MCAP due to a PIK3CA somatic mosaic variant who presented with atrial tachycardia, finally diagnosed as pulmonary arterial hypertension (PAH). Oxygen therapy and sildenafil decreased pulmonary blood pressure and improved atrial tachycardia. Previous studies reported an association between the PI3K/AKT/mTOR pathway and abnormal pulmonary arterial smooth muscle cell proliferation, which may be associated with PAH. PAH should be considered a potentially lethal complication in MCAP patients, even when no structural cardiac abnormalities are identified in the neonatal period.