1. 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 homolog 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.

2. 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.

2.1. 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 dysmorphism, developmental delay, and psychomotor delay features.

Table 1. Clinical details of the affected individuals.

Patient features	V : 1	V : 3	VI : 1	VI : 2
Sex	Male	Female	Male	Female
Consanguinity of parents	First cousin		First cousin
Age (years)	13	7	14	5
Height (cm)	150	117	136	88
Age at first words (months)	20	19	20	19
Age at walking (months)	32	28	33	28
Intellectual disability	Moderate	Moderate	Moderate	Moderate
Microcephaly	+	+	+	+
Neutropenia	+	+	+	+
Hypotonia	-	-	-	+
Motor delay	-	-	+	+
Speech delay	+	+	+	+
Muscular atrophy	-	-	-	+
Insomnia/sleep disturbances	-	-	-	+
Neonatal feeding difficulties	+	+	+	+
Strabismus	+	-	-	+
Wave shape eyelid	+	+	+	+
Long and thick eyelashes	+	+	+	+
Large ear	+	+	+	+
Prominent nose root	+	+	+	+
Prominent upper central incisors	+	+	+	+
Narrow roof mouth	+	+	+	+
Short philtrum	+	+	+	+
Truncal obesity	+	+	+	+
Small hands, feet, and fingers	-	+	-	+

• + Present, - absent.

3. Results

3.2. Exome Sequencing and Follow-Up

Exome sequencing of two affected individuals (V : 3 and VI : 1) in the family revealed a novel homozygous nonsense variant c.8841G > A (NM_017890.5) located in exon 48 out of 62 in the VPS13B gene (Supplementary Figure 1b) at position 8q22.2 (Supplementary Figure 1a). The variant altered the tryptophan amino acid into a premature stop codon p.(W2947 ^∗). The average on-target coverage was 40x, and 24x and 33x at the VPS13B c.8841G > A site for both exomed individuals. Sanger sequencing verified the segregation of this variant in the pedigree (Supplementary Figure 1c). This novel nonsense mutation is absent in gnomAD [20], 1000 genomes [21], GME Variome [22], Kaviar [23], ABraOM [24], AllofUs [25], and TOPMed [26], and bioinformatic tools predict that it significantly damages the function of the VPS13B protein (Table 2). A protein alignment of amino acid sequence 2941 to 2953 of the wild-type human and mutant VPS13B with orthologous proteins of the different species indicated an evolutionary conserved of the C-terminal domain (Figure 2(b)), and the GERP++RS score was 5.98 (Table 2). The variant was classified as pathogenic based on standard guidelines of the American College of Medical Genetics (ACMG) [27].

4. 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 consanguineous families underlying CS have described additional features associated with Cohen syndrome including cerebellar hypoplasia [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 N- and 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.

Conflicts of Interest

The authors declare they have no conflicts of interest.

Authors’ Contributions

Asif Mir and Isabelle Schrauwen contributed equally to this work.