A Novel Variant in VPS13B Underlying Cohen Syndrome

Pathogenic variants in vacuolar protein sorting 13 homolog B (VPS13B) cause Cohen syndrome (CS), a clinically diverse neurodevelopmental disorder. We used whole exome and Sanger sequencing to identify disease-causing variants in a Pakistani family with intellectual disability, microcephaly, facial dysmorphism, neutropenia, truncal obesity, speech delay, motor delay, and insomnia. We identified a novel homozygous nonsense variant c.8841G > A: p.(W2947∗) in VPS13B (NM_017890.5) which segregated with the disease. Sleep disturbances are commonly seen in neurodevelopmental disorders and can exacerbate medical issues if left untreated. We demonstrate that individuals with Cohen syndrome may also be affected by sleep disturbances. In conclusion, we expand the genetic and phenotypic features of Cohen syndrome in the Pakistani population.


Introduction
Cohen syndrome (CS) is a rare heritable autosomal recessive disorder that includes intellectual disability (ID), developmental delay, microcephaly, and hypotonia. Myopia and retinal dystrophy are additional common characteristics, but hypermobility, facial dysmorphism, and a bulbous nasal tip are rarer features [1]. Cohen syndrome has a wide range of clinical characteristics among those who are affected. Additional signs and symptoms in certain people with this syndrome include neutropenia, autistic-like features, and truncal obesity. Affected individuals may also have small hands, feet, and fingers [2]. CS is diagnosed at a frequency of 0.7% in individuals with unexplained intellectual disability [3].
CS is caused by variants in VPS13B (also known as COH1), which codes for vacuolar protein sorting 13 homo-log B. It is a 4022-amino-acid transmembrane protein that is located on chromosome 8 (8q22.2). The protein encoded by this gene is a Golgi-associated peripheral membrane protein involved in Golgi integrity and homeostasis, as well as membrane transport and it is a member of the VPS13 protein family, which is extremely well preserved in eukaryotic cells [4]. Chorea acanthocytosis (OMIM: 200150), rapidly progressive, early-onset autosomal recessive Parkinson's disease (OMIM: 616840), and spinocerebellar ataxia, autosomal recessive 4 (OMIM: 607317) are all possible outcomes of loss of function variants in other VPS13 family members [5].
With the fast advancement of high-throughput sequencing technology, exome sequencing has enabled patients with subtle clinical symptoms to get an early and precise molecular diagnosis, thereby enhancing patient quality of life and simplifying genetic counseling [6]. Due to its massive benefits such as high efficiency, cost, and high accuracy, exome sequencing has been widely used in clinical practice and research [7]. In this study, we investigated a large consanguineous pedigree with ID from Pakistan via exome sequencing.

Methods
This research was performed in accordance with the Declaration of Helsinki. Ethical approval for the study was obtained from the Institutional Review Board (IRB) of the involved institutions, the International Islamic University Islamabad (IIUI/BIBT/FBAS-2022/77) and Columbia University (IRB-AAAS3433). Informed consent was obtained from healthy adult subjects, the parents/legal guardians of minor subjects, and the ID patients in this study to publish the findings of the study.

Clinical Assessment.
A large consanguineous family with ID (MMR-329) of Pashtun ancestry was ascertained in Pakistan (Figure 1(a)). Pedigree information was recorded up to six generations, with a total of seven affected individuals of whom three (IV : 7, IV : 8, and IV : 9) are deceased. Living affected individuals were examined by a local neurologist and psychiatrist (Figure 1(b)). The phenotypic information of all affected members of the family was noted in detail (Table 1) which included age, sex, height, ID, facial dys-morphism, developmental delay, and psychomotor delay features.

Exome
Sequencing. The extraction of genomic DNA was done from the blood of the patients and unaffected members of the family by standard organic methods of phenolchloroform for genetic analysis. DNA samples from two patients (V : 3 and VI : 1) were exome sequenced using the Twist+RefSeq library preparation kit (Twist Bioscience, San Francisco, CA, USA). Barcoded libraries were pooled, and sequencing was performed on the NovaSeq 6000 (Illumina Inc., San Diego, CA, USA) with an average on-target coverage of 40X.

Discussion
CS is a rare autosomal recessive disorder mainly characterized by ID, impaired growth, microcephaly, neutropenia, truncal obesity, and facial dysmorphism, with other additional features including myopia and retinal dystrophy, small fingers, and a bulbous nasal tip [28]. In this study, we observed phenotypic diversity among the affected individuals of the same family which includes motor delay (VI : 1 and VI : 2), strabismus (V : 1 and VI : 2), and hypotonia, muscle atrophy, and insomnia (VI : 2). Furthermore, all affected individuals manifested characteristic CS features including moderate ID, microcephaly, neutropenia, developmental delay, truncal obesity, a bulbous nasal tip, small fingers, and facial dysmorphism. Previous studies in Pakistani   [29], autistic-like features [30], and individuals with a milder form of CS [31]. Moreover, in the present study, we found a previously undescribed feature (insomnia) in CS in the affected individual (VI : 2). This individual has difficulty falling asleep, repeated awakenings with difficulty returning to sleep, or sleep that is nonrestorative or poor in quality. Sleep disturbances such as insomnia are a common feature in many neurodevelopmental disorders (NDDs) [32] such as the Angelman syndrome (AS), autism spectrum disorder (ASD), the Smith-Magenis syndrome (SMS), the Prader-Willi syndrome (PWS), tuberous sclerosis complex (TSC) [33], Fragile X syndrome (FXS), the Williams syndrome (WS), and the Rett syndrome (RTT). Not only are sleep disturbances significantly higher in NDDs than in age-matched unaffected children but they also often last longer as well, such as into adolescence and adulthood [32]. Untreated sleep disorders can aggravate their medical issues, and early interventions may be beneficial to the patient's overall health.
In this study, we report a novel genetic variant in VPS13B [NM_017890.5, c.8841G > A: p.(W2947 * )] in an autosomal recessive consanguineous Pakistani CS family. Over 200 variants have been reported worldwide in multiple domains of VPS13B associated with CS [34]. VPS13B protein has ten transmembrane domains and a potential vacuolar targeting motif, an endoplasmic reticulum retention signal on the C-terminus and two peroxisomal matrix protein targeting signal 2 (PTS2) consensus sequence both on the Nand C-terminus (Figure 2(a)) [35]. Our nonsense variant resides in between the 8th and 9th transmembrane domains of VPS13B, and at this position, the variant is likely targeted via nonsense mRNA-mediated decay (NMD), resulting in no or limited truncated protein expression.
In conclusion, we report a novel nonsense variant in VPS13B associated with CS in a large Pakistani family which displayed phenotypic variability and an expanded phenotype. This study will help facilitate the diagnosis and genetic counseling of families with CS-related features in the Pakistani population.