Bi-allelic ACBD6 variants lead to a neurodevelopmental syndrome with progressive and complex movement disorders.

The acyl-CoA-binding domain-containing protein 6 (ACBD6) is ubiquitously expressed, plays a role in the acylation of lipids and proteins and regulates the N-myristoylation of proteins via N-myristoyltransferase enzymes (NMTs). However, its precise function in cells is still unclear, as is the consequence of ACBD6 defects on human pathophysiology. Using exome sequencing and extensive international data sharing efforts, we identified 45 affected individuals from 28 unrelated families (consanguinity 93%) with bi-allelic pathogenic, predominantly loss-of-function (18/20) variants in ACBD6. We generated zebrafish and Xenopus tropicalis acbd6 knockouts by CRISPR/Cas9 and characterized the role of ACBD6 on protein N-myristoylation with myristic acid alkyne (YnMyr) chemical proteomics in the model organisms and human cells, with the latter also being subjected further to ACBD6 peroxisomal localization studies. The affected individuals (23 males and 22 females), aged 1-50 years, typically present with a complex and progressive disease involving moderate-to-severe global developmental delay/intellectual disability (100%) with significant expressive language impairment (98%), movement disorders (97%), facial dysmorphism (95%) and mild cerebellar ataxia (85%) associated with gait impairment (94%), limb spasticity/hypertonia (76%), oculomotor (71%) and behavioural abnormalities (65%), overweight (59%), microcephaly (39%) and epilepsy (33%). The most conspicuous and common movement disorder was dystonia (94%), frequently leading to early-onset progressive postural deformities (97%), limb dystonia (55%) and cervical dystonia (31%). A jerky tremor in the upper limbs (63%), a mild head tremor (59%), parkinsonism/hypokinesia developing with advancing age (32%) and simple motor and vocal tics were among other frequent movement disorders. Midline brain malformations including corpus callosum abnormalities (70%), hypoplasia/agenesis of the anterior commissure (66%), short midbrain and small inferior cerebellar vermis (38% each) as well as hypertrophy of the clava (24%) were common neuroimaging findings. Acbd6-deficient zebrafish and Xenopus models effectively recapitulated many clinical phenotypes reported in patients including movement disorders, progressive neuromotor impairment, seizures, microcephaly, craniofacial dysmorphism and midbrain defects accompanied by developmental delay with increased mortality over time. Unlike ACBD5, ACBD6 did not show a peroxisomal localization and ACBD6-deficiency was not associated with altered peroxisomal parameters in patient fibroblasts. Significant differences in YnMyr-labelling were observed for 68 co- and 18 post-translationally N-myristoylated proteins in patient-derived fibroblasts. N-myristoylation was similarly affected in acbd6-deficient zebrafish and X. tropicalis models, including Fus, Marcks and Chchd-related proteins implicated in neurological diseases. The present study provides evidence that bi-allelic pathogenic variants in ACBD6 lead to a distinct neurodevelopmental syndrome accompanied by complex and progressive cognitive and movement disorders.

Pentanucleotide Repeat Insertions in RAI1 Cause Benign Adult Familial Myoclonic Epilepsy Type 8.

BACKGROUND: Benign adult familial myoclonic epilepsy (BAFME) is an autosomal dominant disorder characterized by cortical tremors and seizures. Six types of BAFME, all caused by pentanucleotide repeat expansions in different genes, have been reported. However, several other BAFME cases remain with no molecular diagnosis. OBJECTIVES: We aim to characterize clinical features and identify the mutation causing BAFME in a large Malian family with 10 affected members. METHODS: Long-read whole genome sequencing, repeat-primed polymerase chain reaction and RNA studies were performed. RESULTS: We identified TTTTA repeat expansions and TTTCA repeat insertions in intron 4 of the RAI1 gene that co-segregated with disease status in this family. TTTCA repeats were absent in 200 Malian controls. In the affected individuals, we found a read with only nine TTTCA repeat units and somatic instability. The RAI1 repeat expansions cause the only BAFME type in which the disease-causing repeats are in a gene associated with a monogenic disorder in the haploinsufficiency state (ie, Smith-Magenis syndrome [SMS]). Nevertheless, none of the Malian patients exhibited symptoms related to SMS. Moreover, leukocyte RNA levels of RAI1 in six Malian BAFME patients were no different from controls. CONCLUSIONS: These findings establish a new type of BAFME, BAFME8, in an African family and suggest that haploinsufficiency is unlikely to be the main pathomechanism of BAFME. © 2023 International Parkinson and Movement Disorder Society. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Mutations in Kinesin family member 6 reveal specific role in ependymal cell ciliogenesis and human neurological development.

Cerebrospinal fluid flow is crucial for neurodevelopment and homeostasis of the ventricular system of the brain, with localized flow being established by the polarized beating of the ependymal cell (EC) cilia. Here, we report a homozygous one base-pair deletion, c.1193delT (p.Leu398Glnfs*2), in the Kinesin Family Member 6 (KIF6) gene in a child displaying neurodevelopmental defects and intellectual disability. To test the pathogenicity of this novel human KIF6 mutation we engineered an analogous C-terminal truncating mutation in mouse. These mutant mice display severe, postnatal-onset hydrocephalus. We generated a Kif6-LacZ transgenic mouse strain and report expression specifically and uniquely within the ependymal cells (ECs) of the brain, without labeling other multiciliated mouse tissues. Analysis of Kif6 mutant mice with scanning electron microscopy (SEM) and immunofluorescence (IF) revealed specific defects in the formation of EC cilia, without obvious effect of cilia of other multiciliated tissues. Dilation of the ventricular system and defects in the formation of EC cilia were also observed in adult kif6 mutant zebrafish. Finally, we report Kif6-GFP localization at the axoneme and basal bodies of multi-ciliated cells (MCCs) of the mucociliary Xenopus epidermis. Overall, this work describes the first clinically-defined KIF6 homozygous null mutation in human and defines KIF6 as a conserved mediator of neurological development with a specific role for EC ciliogenesis in vertebrates.

TTTCA repeat insertions in an intron of YEATS2 in benign adult familial myoclonic epilepsy type 4.

Epilepsy is a common neurological disorder and identification of its causes is important for a better understanding of its pathogenesis. We previously studied a Thai family with a type of epilepsy, benign adult familial myoclonic epilepsy type 4 (BAFME4), and localized its gene to chromosome 3q26.32-q28. Here, we used single-molecule real-time sequencing and found expansions of TTTTA and insertions of TTTCA repeats in intron 1 of YEATS2 in one affected member of the family. Of all the available members in the family-comprising 13 affected and eight unaffected-repeat-primed PCR and long-range PCR revealed the co-segregation of the TTTCA repeat insertions with the TTTTA repeat expansions and the disease status. For 1116 Thai control subjects, none were found to harbour the TTTCA repeats while four had the TTTTA repeat expansions. Therefore, our findings suggest that BAFME4 is caused by the insertions of the intronic TTTCA repeats in YEATS2. Interestingly, all four types of BAFMEs for which underlying genes have been found (BAFMEs 1, 4, 6 and 7) are caused by the same molecular pathology, suggesting that the insertions of non-coding TTTCA repeats are involved in their pathogenesis.

Widespread and debilitating hemangiomas in a patient with enchondromatosis and D-2-hydroxyglutaric aciduria.

Metaphyseal chondromatosis with D-2-hydroxyglutaric aciduria (MC-HGA) (OMIM 614875) is a severe chondrodysplasia combined with a urinary excretion of D-2-hydroxyglutaric acid. Here, we reported the tenth case of this disease. A 15-year-old boy had symmetric radiolulencies in the metaphyses of the long bones suggesting enchondromatosis and platyspondyly. Remarkably, he manifested widespread cavernous hemangiomas including scalp, lips, tongue, larynx, and prepuce, with the onset of 3 years of age. Hemangiomas at the larynx had caused dyspnea and those in the oral cavity led to recurrent bleeding, requiring several surgical removals. These multiple and debilitating hemangiomas have never been previously reported in patients with MC-HGA. Mutation analyses including Sanger sequencing of genes involving in enchondromatosis and the metabolic pathway of D-2-hydroxyglutarate including PTHR1, D2HGDH, HOT, and IDH1, as well as whole-exome sequencing for proband-parent trio analysis and paired blood versus hemangioma studies showed no pathogenic variants. In summary, we reported the tenth patient with MC-HGA who manifested widespread and debilitating hemangiomas in several organs, expanding the clinical spectrum of MC-HGA.

Delayed diagnosis in a house of correction: Smith-Magenis syndrome due to a de novo nonsense RAI1 variant.

We report a 25-year-old female confirmed to have Smith-Magenis syndrome (SMS) due to a de novo RAI1 variant. Her past history is significant for developmental and intellectual delay, early and escalating maladaptive behaviors, and features consistent with significant sleep disturbance, the etiology of which was not confirmed for over two decades. The diagnosis of SMS was initially suspected in 1998 (at age 12 years), but that was 5 years before the initial report of RAI1 variants as causative of the SMS phenotype; cytogenetic fluorescence in situ hybridization studies failed to confirm an interstitial deletion of 17p11.2. Re-evaluation for suspected SMS was pursued with RAI1 sequencing analysis in response to urgent parental concerns of escalating behaviors and aggression with subsequent incarceration of the subject for assault of a health professional. Genetic analysis revealed a de novo RAI1 (NM_030665.3) nonsense variant, c.5536C>T; p.Q1846X. This case illustrates the importance of confirming the SMS diagnosis, which is associated with cognitive and functional impairment, as well as significant psychiatric co-morbidities and behavioral problems. The diagnosis was particularly relevant to the legal discussion and determination of her competence to stand trial. As other similar cases may exist, this report will help to increase awareness of the possibility of a very late diagnosis of SMS, with the need for re-evaluation of individuals suspected to have SMS who were initially evaluated prior to the identification of the RAI1 gene. © 2016 Wiley Periodicals, Inc.

Splicing analysis of CYP11B1 mutation in a family affected with 11ß-hydroxylase deficiency: case report.

BACKGROUND: Congenital adrenal hyperplasia (CAH) due to steroid 11ß-hydroxylase deficiency (11ß-OHD) is a rare form of CAH associated with low renin hypertension, hypokalemia, hyperandrogenemia and ambiguous genitalia in affected females. Herein we describe the clinical, hormonal and molecular characteristics of two Uzbekistan siblings with 11ß-OHD and analyze the effects of a splicing mutation. CASE PRESENTATION: A 46,XX girl presented with genital ambiguity and low renin hypertension; her 46,XY brother presented with precocious puberty. Hormonal studies suggested 11ß-OHD. Mutation analysis was performed by PCR followed by Sanger sequencing of the entire coding regions and their flanking introns of the CYP11B1 gene. Mutation analysis showed that both patients were compound heterozygous for IVS7 + 1G > A, and c.421C > T. Although the identified mutations have been previously described, this is, to our knowledge, the first report of these mutations in compound heterozygotes. A minigene assay was used to determine the effects of the splicing mutation. The constructs containing either the wild-type or the splice-site mutant CYP11B1 genomic DNA of exons-introns 6-9 were transfected into COS-7 cells; subsequently, RNA splicing was assessed by reversed transcribed-PCR of CYP11B1 complementary DNA. The minigene assay revealed that the IVS7 + 1G > A mutation resulted in two shorter incorrectly spliced products; one skipping the exon 7 and the other skipping the exons 7-8. The c.421C > T mutation leads to the introduction of a premature stop codon at residue 141 (p.R141X). These mutations are expected to code non-functional proteins. CONCLUSION: Compound heterozygous mutations (IVS7 + 1G > A and p.R141X) in the CYP11B1 gene were found to cause 11ß-OHD. The IVS7 + 1G > A mutation causes aberrant splicing of CYP11B1 leading to exon skipping. This finding could facilitate the future novel therapies targeted on splicing modulation to treat human disease.

A Frameshift Mutation in PEN-2 Causes Familial Comedones Syndrome.

BACKGROUND: Familial comedones without dyskeratosis are a rare autosomal dominant skin disorder, characterized by the occurrence of comedones that are distributed all over the body with specific features. We have previously reported two Thai families with familial comedones with expanded phenotypic spectrum. However, its genetic defect and pathogenesis remain unknown. OBJECTIVE: To explore the molecular defect causing familial comedones. METHODS: Whole-genome linkage analysis and whole-exome sequencing in family I were performed. RESULTS: We identified a heterozygous one-base pair insertion, c.84_85insT (p. L28FfsX93) in PEN-2, located within the linked region on chromosome 19. PCR-Sanger sequencing confirmed the identified mutation. The mutation segregated with the disease phenotype in family I and was fully penetrant. This similar mutation was also present in the unrelated affected individual from family II. Quantitative PCR revealed increased mRNA expression of PEN-2 in leukocytes of affected individuals. CONCLUSION: We for the first time identify PEN-2 as the causative gene of familial comedones.

Neurodevelopmental Disorder, Obesity, Pancytopenia, Diabetes Mellitus, Cirrhosis, and Renal Failure in ACBD6-Associated Syndrome: A Case Report.

Objectives: Neurodevelopmental disorders (NDDs) are a group of conditions that are clinically and etiologically heterogeneous. Biallelic variants in ACBD6 were previously reported in 7 patients with NDDs. Unfortunately, their clinical information remains very limited with descriptions of only their neurologic and craniofacial features. The purpose of this report is to expand the clinical phenotype of the ACBD6-associated NDDs. Methods: We identified 2 Thai siblings with NDDs. Clinical and radiologic features of the proband were described. The affected siblings and parents underwent whole-exome sequencing and PCR-Sanger sequencing. Results: Clinical manifestations that have never been previously reported include morbid obesity, pancytopenia with severe infections, diabetes mellitus, cirrhosis, and renal failure, leading to deaths in their early 30s. Molecular studies identified a novel homozygous 1 base-pair duplication (c.360dup; p.Leu121Thrfs*27) in the ACBD6 gene. Discussion: This study reported 1 novel single base-pair duplication, expanding the mutational spectrum, and described the clinical features establishing the entity of ACBD6-associated NDDs.

Long-read Nanopore sequencing identified D4Z4 contractions in patients with facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is a genetic muscle disorder caused by abnormal expression of the DUX4 protein, commonly resulting from a contraction of D4Z4 repeat units with the presence of a polyadenylation (polyA) signal. More than 10 units of the D4Z4 repeat, with a length of 3.3 kb per unit, are typically required to silence DUX4 expression. Consequently, molecular diagnosis of FSHD is challenging. We used Oxford Nanopore technology to perform whole-genome sequencing of seven unrelated patients with FSHD, their six unaffected parents, and 10 unaffected controls. All seven patients were successfully identified to harbor one to five D4Z4 repeat units and the polyA signal, whereas none of the 16 unaffected individuals met the molecular diagnostic criteria. Our newly developed method provides a straightforward and powerful molecular diagnostic tool for FSHD.

Primary hyperoxaluria type 1 and brachydactyly mental retardation syndrome caused by a novel mutation in AGXT and a terminal deletion of chromosome 2.

Primary hyperoxaluria type 1 (PH1) is an autosomal recessive disorder caused by mutations in the alanine:glyoxylate aminotransferase (AGXT) gene, located on chromosome 2q37. Mutant AGXT leads to excess production and excretion of oxalate, resulting in accumulation of calcium oxalate in the kidney, and progressive loss of renal function. Brachydactyly mental retardation syndrome (BDMR) is an autosomal dominant disorder, caused by haploinsufficiency of histone deacetylase 4 (HDAC4), also on chromosome 2q37. It is characterized by skeletal abnormalities and developmental delay. Here, we report on a girl who had phenotypes of both PH1 and BDMR. PCR-sequencing of the coding regions of AGXT showed a novel missense mutation, c.32C>G (p.Pro11Arg) inherited from her mother. Functional analyses demonstrated that it reduced the enzymatic activity to 31% of the wild-type and redirected some percentage of the enzyme away from the peroxisome. Microsatellite and array-CGH analyses indicated that the proband had a paternal de novo telomeric deletion of chromosome 2q, which included HDAC4. To our knowledge, this is the first report of PH1 and BDMR, with a novel AGXT mutation and a de novo telomeric deletion of chromosome 2q.

Long-read Amplicon Sequencing of the CYP21A2 in 48 Thai Patients With Steroid 21-Hydroxylase Deficiency.

CONTEXT: Congenital adrenal hyperplasia is most commonly caused by 21-hydroxylase deficiency (21-OHD), an autosomal recessive disorder resulting from biallelic pathogenic variants (PVs) in CYP21A2. With a highly homologous pseudogene and various types of single nucleotide and complex structural variants, identification of PVs in CYP21A2 has been challenging. OBJECTIVE: To leverage long-read next-generation sequencing combined with locus-specific polymerase chain reaction (PCR) to detect PVs in CYP21A2 and to determine its diagnostic yield in patients with 21-OHD. METHODS: Forty-eight Thai patients with 21-OHD comprising 38 sporadic cases and 5 pairs of siblings were enrolled. Two previously described locus-specific PCR methods were performed. Amplicons were subject to long-read sequencing. RESULTS: Ninety-six PVs in CYP21A2 in the 48 patients were successfully identified. The combined techniques were able to detect 26 structural chimeric variants (27%; 26/96) in 22 patients with 18 having monoallelic and 4 having biallelic chimeras. The remaining PVs were pseudogene-derived mutations (63%; 60/96), entire gene deletions (2%; 2/96), missense variants (3%; 3/96), a splice-site variant (2%; 2/96), frameshift variants (2%; 2/96), and a nonsense variant (1%; 1/96). Notably, a splice-site variant, IVS7 + 1G > T, which was identified in a pair of siblings, has not previously been reported. CONCLUSIONS: Our approach exploiting locus-specific PCR and long-read DNA sequencing has a 100% diagnostic yield for our cohort of 48 patients with 21-OHD.

Founder effect of the TTTCA repeat insertions in SAMD12 causing BAFME1.

Benign adult familial myoclonic epilepsy type 1 (BAFME1) in several Japanese and Chinese families has recently been found to be caused by pentanucleotide repeat expansions in SAMD12. We identified a Thai family with six members affected with BAFME. Microsatellite studies suggested a linkage to the BAFME1 region on chromosome 8q24. Subsequently, long-read whole-genome sequencing showed the (TTTTA)446(TTTCA)149 in intron 4 of SAMD12 in an affected member. Repeat-primed PCR and long-range PCR revealed that the pentanucleotide repeat expansions segregated with the disease status. Our Thai family is the first non-Japanese and non-Chinese family with BAFME1. SNP array showed that the aberrant repeats had the same haplotype as those previously determined in Japanese and Chinese patients suggesting a common ancestry. The variant is estimated to arise ~12,000 years ago.

Three novel mutations of the IRF6 gene with one associated with an unusual feature in Van der Woude syndrome.

Van der Woude syndrome (VWS) is a dominantly inherited disorder characterized by cleft lip with or without cleft palate and lip pits. It remains the most common syndromic form of oral clefts. Mutations in the interferon regulatory factor 6 (IRF6) gene have been identified in patients with VWS. We reported three unrelated families with lower lip anomalies. Two had lower lip pits, a cardinal sign of VWS, but the other had a heart-shaped mass on lower lip without pits, oral clefts, or hypodontia. This isolated anomaly has not been previously observed in VWS. We performed mutation analysis by PCR-sequencing the entire coding region of the IRF6 gene. Three potentially pathogenic mutations, c.145C>T (p.Q49X), c.171T>G (p.F57L), and 1306C>G (p.L436V) were successfully identified. All the missense mutations were not detected in 100 unaffected ethnic-matched control chromosomes and have never been previously reported. The p.Q49X and p.F57L mutations were located in the highly conserved DNA binding domain while the p.L436V was located at the carboxy-terminal region. This study reported an undescribed clinical feature of VWS and three novel mutations, expanding the phenotypic spectrum of VWS and mutational spectrum of IRF6.

MBTPS2 mutations cause defective regulated intramembrane proteolysis in X-linked osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a collagen-related bone dysplasia. We identified an X-linked recessive form of OI caused by defects in MBTPS2, which encodes site-2 metalloprotease (S2P). MBTPS2 missense mutations in two independent kindreds with moderate/severe OI cause substitutions at highly conserved S2P residues. Mutant S2P has normal stability, but impaired functioning in regulated intramembrane proteolysis (RIP) of OASIS, ATF6 and SREBP transcription factors, consistent with decreased proband secretion of type I collagen. Further, hydroxylation of the collagen lysine residue (K87) critical for crosslinking is reduced in proband bone tissue, consistent with decreased lysyl hydroxylase 1 in proband osteoblasts. Reduced collagen crosslinks presumptively undermine bone strength. Also, proband osteoblasts have broadly defective differentiation. These mutations provide evidence that RIP plays a fundamental role in normal bone development.

A newly identified locus for benign adult familial myoclonic epilepsy on chromosome 3q26.32-3q28.

Benign Adult Familial Myoclonic Epilepsy (BAFME) is an autosomal dominant disorder characterized by adult-onset cortical tremor or action myoclonus predominantly in the upper limbs, and generalized seizures. We investigated a Thai BAFME family. Clinical and electrophysiological studies revealed that 13 were affected with BAFME. There were a total of 24 individuals studied. Genetic analysis by genome-wide linkage study (GWLS) was performed using 400 microsatellite markers and excluded linkage of the previous BAFME loci, 8q23.3-q24.1, and 2p11.1-q12.2. GWLS showed that the disease-associated region in our Thai family was linked to a newly identified locus on chromosome 3q26.32-3q28. This locus represents the fourth chromosomal region for BAFME.

Two novel CTNS mutations in cystinosis patients in Thailand.

Cystinosis is an autosomal recessive disorder characterized by defective transport of cystine across the lysosomal membrane and resulting in renal, ophthalmic, and other organ abnormalities. Mutations in the CTNS gene cause a deficiency of the transport protein, cystinosin. We performed mutation analysis of CTNS in six cystinosis patients from four families in Thailand. Using PCR sequencing of the entire coding regions, we identified all eight mutant alleles, including two mutations, p.G309D and p.Q284X, that have not been previously reported. This study expands the mutational and population spectrum of nephropathic cystinosis.