Molecular insight into CREBBP and TANGO2 variants causing intellectual disability.

BACKGROUND: Intellectual disability (ID) can be associated with different syndromes such as Rubinstein-Taybi syndrome (RSTS) and can also be related to conditions such as metabolic encephalomyopathic crises, recurrent,with rhabdomyolysis, cardiac arrhythmias and neurodegeneration. Rare congenital RSTS1 (OMIM 180849) is characterized by mental and growth retardation, significant and duplicated distal phalanges of thumbs and halluces, facial dysmorphisms, and an elevated risk of malignancies. Microdeletions and point mutations in the CREB-binding protein (CREBBP) gene, located at 16p13.3, have been reported to cause RSTS. By contrast, TANGO2-related metabolic encephalopathy and arrhythmia (TRMEA) is a rare metabolic condition that causes repeated metabolic crises, hypoglycemia, lactic acidosis, rhabdomyolysis, arrhythmias and encephalopathy with cognitive decline. Clinicians need more clinical and genetic evidence to detect and comprehend the phenotypic spectrum of this disorder. METHODS: Exome sequencing was used to identify the disease-causing variants in two affected families A and B from District Kohat and District Karak, Khyber Pakhtunkhwa. Affected individuals from both families presented symptoms of ID, developmental delay and behavioral abnormalities. The validation and co-segregation analysis of the filtered variant was carried out using Sanger sequencing. RESULTS: In the present study, two families (A and B) exhibiting various forms of IDs were enrolled. In Family A, exome sequencing revealed a novel missense variant (NM 004380.3: c.4571A>G; NP_004371.2: p.Lys1524Arg) in the CREBBP gene, whereas, in Family B, a splice site variant (NM 152906.7: c.605 + 1G>A) in the TANGO2 gene was identified. Sanger sequencing of both variants confirmed their segregation with ID in both families. The in silico tools verified the aberrant changes in the CREBBP protein structure. Wild-type and mutant CREBBP protein structures were superimposed and conformational changes were observed likely altering the protein function. CONCLUSIONS: RSTS and TRMEA are exceedingly rare disorders for which specific clinical characteristics have been clearly established, but more investigations are underway and required. Multicenter studies are needed to increase our understanding of the clinical phenotypes, mainly showing the genotype-phenotype associations.

Structural and functional implications of SLC13A3 and SLC9A6 mutations: an in silico approach to understanding intellectual disability.

BACKGROUND: Intellectual disability (ID) is a condition that varies widely in both its clinical presentation and its genetic underpinnings. It significantly impacts patients' learning capacities and lowers their IQ below 70. The solute carrier (SLC) family is the most abundant class of transmembrane transporters and is responsible for the translocation of various substances across cell membranes, including nutrients, ions, metabolites, and medicines. The SLC13A3 gene encodes a plasma membrane-localized Na+/dicarboxylate cotransporter 3 (NaDC3) primarily expressed in the kidney, astrocytes, and the choroid plexus. In addition to three Na + ions, it brings four to six carbon dicarboxylates into the cytosol. Recently, it was discovered that patients with acute reversible leukoencephalopathy and a-ketoglutarate accumulation (ARLIAK) carry pathogenic mutations in the SLC13A3 gene, and the X-linked neurodevelopmental condition Christianson Syndrome is caused by mutations in the SLC9A6 gene, which encodes the recycling endosomal alkali cation/proton exchanger NHE6, also called sodium-hydrogen exchanger-6. As a result, there are severe impairments in the patient's mental capacity, physical skills, and adaptive behavior. METHODS AND RESULTS: Two Pakistani families (A and B) with autosomal recessive and X-linked intellectual disorders were clinically evaluated, and two novel disease-causing variants in the SLC13A3 gene (NM 022829.5) and the SLC9A6 gene (NM 001042537.2) were identified using whole exome sequencing. Family-A segregated a novel homozygous missense variant (c.1478 C > T; p. Pro493Leu) in the exon-11 of the SLC13A3 gene. At the same time, family-B segregated a novel missense variant (c.1342G > A; p.Gly448Arg) in the exon-10 of the SLC9A6 gene. By integrating computational approaches, our findings provided insights into the molecular mechanisms underlying the development of ID in individuals with SLC13A3 and SLC9A6 mutations. CONCLUSION: We have utilized in-silico tools in the current study to examine the deleterious effects of the identified variants, which carry the potential to understand the genotype-phenotype relationships in neurodevelopmental disorders.

Autosomal recessive variants c.953A>C and c.97-1G>C in NSUN2 causing intellectual disability: a molecular dynamics simulation study of loss-of-function mechanisms.

Introduction: Intellectual disability (ID) is a clinically and genetically heterogeneous disorder. It drastically affects the learning capabilities of patients and eventually reduces their IQ level below 70. Methods: The current genetic study ascertained two consanguineous Pakistani families suffering from autosomal recessive intellectual developmental disorder-5 (MRT5). We have used exome sequencing followed by Sanger sequencing to identify the disease-causing variants. Results and discussion: Genetic analysis using whole exome sequencing in these families identified two novel mutations in the NSUN2 (NM_017755.5). Family-A segregated a novel missense variant c.953A>C; p.Tyr318Ser in exon-9 of the NSUN2. The variant substituted an amino acid Tyr318, highly conserved among different animal species and located in the functional domain of NSUN2 known as "SAM-dependent methyltransferase RsmB/NOP2-type". Whereas in family B, we identified a novel splice site variant c.97-1G>C that affects the splice acceptor site of NSUN2. The identified splice variant (c.97-1G>C) was predicted to result in the skipping of exon-2, which would lead to a frameshift followed by a premature stop codon (p. His86Profs*16). Furthermore, it could result in the termination of translation and synthesis of dysfunctional protein, most likely leading to nonsense-mediated decay. The dynamic consequences of NSUN2 missense variant was further explored together with wildtype through molecular dynamic simulations, which uncovered the disruption of NSUN2 function due to a gain in structural flexibility. The present molecular genetic study further extends the mutational spectrum of NSUN2 to be involved in ID and its genetic heterogeneity in the Pakistani population.

Biallelic Variants in Seven Different Genes Associated with Clinically Suspected Bardet-Biedl Syndrome.

Bardet-Biedl syndrome (BBS) is a rare clinically and genetically heterogeneous autosomal recessive multi-systemic disorder with 22 known genes. The primary clinical and diagnostic features include six different hallmarks, such as rod-cone dystrophy, learning difficulties, renal abnormalities, male hypogonadism, post-axial polydactyly, and obesity. Here, we report nine consanguineous families and a non-consanguineous family with several affected individuals presenting typical clinical features of BBS. In the present study, 10 BBS Pakistani families were subjected to whole exome sequencing (WES), which revealed novel/recurrent gene variants, including a homozygous nonsense mutation (c.94C>T; p.Gln32Ter) in the IFT27 (NM_006860.5) gene in family A, a homozygous nonsense mutation (c.160A>T; p.Lys54Ter) in the BBIP1 (NM_001195306.1) gene in family B, a homozygous nonsense variant (c.720C>A; p.Cys240Ter) in the WDPCP (NM_015910.7) in family C, a homozygous nonsense variant (c.505A>T; p.Lys169Ter) in the LZTFL1 (NM_020347.4) in family D, pathogenic homozygous 1 bp deletion (c.775delA; p.Thr259Leufs*21) in the MKKS/BBS5 (NM_170784.3) gene in family E, a pathogenic homozygous missense variant (c.1339G>A; p.Ala447Thr) in BBS1 (NM_024649.4) in families F and G, a pathogenic homozygous donor splice site variant (c.951+1G>A; p?) in BBS1 (NM_024649.4) in family H, a pathogenic bi-allelic nonsense variant in MKKS (NM_170784.3) (c.119C>G; p.Ser40*) in family I, and homozygous pathogenic frameshift variants (c.196delA; p.Arg66Glufs*12) in BBS5 (NM_152384.3) in family J. Our findings extend the mutation and phenotypic spectrum of four different types of ciliopathies causing BBS and also support the importance of these genes in the development of multi-systemic human genetic disorders.

Biallelic mutations in FLG, TGM1, and STS genes segregated with different types of ichthyoses in eight families of Pakistani origin.

BACKGROUND: Congenital ichthyosis is a diverse group of keratinization disorders associated with generalized scaling of skin of varying severity. The non-syndromic forms of congenital ichthyosis are further grouped into common ichthyosis (ichthyosis vulgaris and X-linked ichthyosis), autosomal recessive congenital ichthyosis, and keratopathic ichthyosis. OBJECTIVE: To identify sequence variants involved in different forms of hereditary ichthyoses. METHODS: We studied eight families with different types of ichthyosis including four families with autosomal recessive congenital ichthyosis and four families with common ichthyosis. Whole exome sequencing and PCR based genotyping was carried out to find out the molecular basis of disease. RESULTS: In one family, a novel duplication sequence variant NM_002016.2:c.2767dupT; NP_002007.1:p.Ser923PhefsTer2 was identified in FLG gene; in four families a previously reported nonsense sequence variant NM_000359.3:c.232C>T; NP_002007.1:p.Arg78Ter was identified in TGM1 gene, while, in three families of X-linked recessive ichthyosis, the whole STS gene (NM_001320752.2; NP_001307681.2) regions were deleted. STUDY LIMITATION: Gene expression studies have not been performed that would have strengthened the findings of computational analysis. CONCLUSION: This study highlights the significance of the c.232C>T variant in the TGM1 gene as a possible founder mutation, complete STS gene deletion as reported previously in Pakistani population, while novel sequence variant in the FLG gene expands the spectrum of variations in this gene. These findings may be used for genetic counseling of the studied families.

Biallelic mutations in the LPAR6 gene causing autosomal recessive wooly hair/hypotrichosis phenotype in five Pakistani families.

BACKGROUND: Autosomal recessive wooly hair/hypotrichosis is an inherited disorder of hair characterized by less dense, short, and tightly curled hair on the scalp and sometimes less dense to complete absence of eyebrows and eyelashes. Autosomal recessive wooly hair/hypotrichosis phenotypes are mostly associated with pathogenic sequence variants in LIPH and LPAR6 genes. METHODS: To find out the molecular basis of the disease, five families with autosomal recessive wooly hair/hypotrichosis were recruited for genetic analysis. Direct Sanger sequencing of LIPH and LPAR6 genes was carried out using BigDye chain termination chemistry. P2RY5 protein homology models were developed to study the effect of mutation on protein structure in a family having novel mutation. RESULTS: Sanger sequencing revealed a novel homozygous missense mutation (c.47A>T) in the LPAR6 gene in family A, while recurrent mutation (c.436G>A) was detected in the rest of the four families (B-E). Protein homology models for both native and mutant P2RY5 protein were developed to study the difference in subtle structural features because of Lys16Met (K16M) mutation. We observed that P2RY5K16M mutation results decrease in the number of ionic interactions detrimental to the protein stability. Protein modeling studies revealed that the novel mutation identified here decreased the number of ionic interactions by affecting physicochemical parameters of the protein, leading to an overall decrease in protein stability with no major secondary structural changes. CONCLUSION: The molecular analysis further confirms the frequent involvement of LPAR6 in autosomal recessive wooly hair/hypotrichosis, while the bioinformatic study revealed that the missense mutation destabilizes the overall structure of P2RY5 protein.

Pakistan Genetic Mutation Database (PGMD); A centralized Pakistani mutome data source.

The development and advancement of next generation sequencing have not only sped up the process of identifying rare variants, but have also enabled scientists to explore all variants in a single individual. The Pakistani population has a high ratio of first degree consanguinity, which is why it is a rich source for various kinds of genetic disorders. Due to the heterogeneous composition of Pakistani population, the likelihood of genetic heterogeneity for each disorder is high. Therefore, the compilation and organization of such vast genetic data is necessary to facilitate access for analysis and interpretation to researchers and medical geneticists. The increased research on Pakistani ethnic families for disease gene identification has revealed many mutations, which has led us to develop a Pakistani mutome database entitled "Pakistan Genetic Mutation Database (PGMD)". In PGMD, the medico-genetic information about diseases are mainly compiled into Syndromic and Non-syndromic disorders. It is a public database, which can be freely accessed from http://www.pakmutation.com. At present, we have registered more than 1000 mutations, reported in about 130 different kinds of genetic disorders. Practically, PGMD will assist researchers, clinicians, and geneticists in genetic counseling and screening of population-specific mutations, which will also aid in personalized healthcare.