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.

Homozygosity mapping coupled with whole-exome sequencing and protein modelling identified a novel missense mutation in GUCY2D in a consanguineous Pakistani family with Leber congenital amaurosis.

Leber congenital amaurosis (LCA) is a rare form of early onset vision loss or blindness due to retinal dystrophy. This condition is characterized by early vision loss, nystagmus and severe retinal dysfunction. To date, genetic studies have reported 19 genes to be associated with autosomal recessive LCA, most of which are involved in the retinal morphology and the physiology of the phototransduction pathway. In the current study, a large consanguineous family segregating congenital blindness was ascertained from the Dera Ismail Khan region of Pakistan. Genetic analysis was performed through genomewide SNP genotyping (for homozygosity-by-descent mapping), whole-exome sequencing (for mutation identification) and Sanger sequencing (for segregation analysis). In silico structural predictions were performed through SWISS-Model (structure prediction) and ClusPro (molecular docking). Molecular investigation of the present LCA family identified a novel homozygous missense mutation p.Asp306Val in GUCY2D gene (NM_000180.3:c.917A>T). In silico structural modelling and interaction studies predicted significant changes in protein folding and interacting residues. The present molecular genetic study further extends the mutational spectrum of GUCY2D in LCA, and its genetic heterogeneity in the Pakistani population. The findings of the computational studies on protein structure and interaction profile predicted pathogenic consequences of p.Asp306Val on GUCY2D function.

Homozygous variants of EDAR underlying hypohidrotic ectodermal dysplasia in three consanguineous families.

BACKGROUND: Hypohidrotic ectodermal dysplasia (HED) is a congenital anomaly characterized by hypohydrosis, hypotrichosis and hypodontia. Mutations in at least four genes (EDAR, EDARADD, WNT10A, TRAF6) have been reported to cause both autosomal recessive and autosomal dominant forms of HED. Mutations in two other genes (EDA and IKBKG) have been reported to cause X-linked HED. OBJECTIVES: To clinically characterize three consanguineous families (A-C) segregating with autosomal recessive HED and identify possible disease-causing variants of EDAR and EDARADD genes. MATERIALS AND METHODS: The genes, EDAR and EDARADD, were sequenced in Family A and C, and exome sequencing was performed in Family B. Additionally, in Family A and C, the effect of the identified variants was examined by analysis of EDAR mRNA, extracted from hair follicles from both affected and unaffected members. RESULTS: Sequence analysis revealed three possible disease-causing EDAR variants including a novel splice acceptor site variant (IVS3-1G > A) in Family A and two previously reported mutations (p.[Ala26Val], p.[Arg25*]) in the two other families. Previously, the nonsense variant p.(Arg25*) was reported only in the heterozygous state. Analysis of the RNA, extracted from hair follicles, revealed skipping of a downstream exon in EDAR and complete degradation of EDAR mRNA in affected members in family A and C, respectively. Computational modelling validated the pathogenic effect of the two variants identified in Family B and C. CONCLUSION: The three variants reported here expand the spectrum of EDAR mutations associated with HED which may further facilitate genetic counselling of families segregating with similar disorders in the Pakistani population.

Association of sequence variants in frizzled-6 with autosomal recessive nail dysplasia (NDNC-10) in Pashtun families.

Primitive epidermis develops the nail apparatus. Nails have a strong and inflexible nail plate at the end of each digit. Very few genes responsible for causing nonsyndromic form of nail dysplasia have been reported. In the current study, peripheral blood samples were collectedfrom three unaffected individuals and four affectedindividuals of Family A, while blood from two affected and three unaffected individuals were taken of Family B. Genotyping in both the families was performed using highly polymorphic short tandem repeat microsatellite markers. Sanger sequence of the FZD6 gene was performed and analysed for segregation analysis. A comparative modelling approach was used to predict the three-dimensional structures of FZD-6 protein using Modeller 4. Linkage analysis mapped a disease locus on chromosome 8q22.3, harbouring FZD6. Targeted Sanger sequencing of all the coding exons of FZD6 revealed a nonsense sequence variant in pedigree A, whereas a missense sequence variant in pedigree B. Finding and literature indicates the disease spectrum of Pakistani population with claw-shaped nail dysplasia, particularly in families of Pashtun origin.

Deleterious Variants in WNT10A, EDAR, and EDA Causing Isolated and Syndromic Tooth Agenesis: A Structural Perspective from Molecular Dynamics Simulations.

The dental abnormalities are the typical features of many ectodermal dysplasias along with congenital malformations of nails, skin, hair, and sweat glands. However, several reports of non-syndromic/isolated tooth agenesis have also been found in the literature. The characteristic features of hypohidrotic ectodermal dysplasia (HED) comprise of hypodontia/oligodontia, along with hypohidrosis/anhidrosis, and hypotrichosis. Pathogenic variants in EDA, EDAR, EDARADD, and TRAF6, cause the phenotypic expression of HED. Genetic alterations in EDA and WNT10A cause particularly non-syndromic/isolated oligodontia. In the current project, we recruited 57 patients of 17 genetic pedigrees (A-Q) from different geographic regions of the world, including Pakistan, Egypt, Saudi Arabia, and Syria. The molecular investigation of different syndromic and non-syndromic dental conditions, including hypodontia, oligodontia, generalized odontodysplasia, and dental crowding was carried out by using exome and Sanger sequencing. We have identified a novel missense variant (c.311G>A; p.Arg104His) in WNT10A in three oligodontia patients of family A, two novel sequence variants (c.207delinsTT, p.Gly70Trpfs*25 and c.1300T>G; p.Try434Gly) in EDAR in three patients of family B and four patients of family C, respectively. To better understand the structural and functional consequences of missense variants in WNT10A and EDAR on the stability of the proteins, we have performed extensive molecular dynamic (MD) simulations. We have also identified three previously reported pathogenic variants (c.1076T>C; p.Met359Thr), (c.1133C>T; p.Thr378Met) and (c.594_595insC; Gly201Argfs*39) in EDA in family D (four patients), E (two patients) and F (one patient), correspondingly. Presently, our data explain the genetic cause of 18 syndromic and non-syndromic tooth agenesis patients in six autosomal recessive and X-linked pedigrees (A-F), which expand the mutational spectrum of these unique clinical manifestations.

A novel pathogenic missense variant in CNNM4 underlying Jalili syndrome: Insights from molecular dynamics simulations.

BACKGROUND: Jalili syndrome (JS) is a rare cone-rod dystrophy (CRD) associated with amelogenesis imperfecta (AI). The first clinical presentation of JS patients was published in 1988 by Jalili and Smith. Pathogenic mutations in the Cyclin and CBS Domain Divalent Metal Cation Transport Mediator 4 (CNNM4) magnesium transporter protein have been reported as the leading cause of this anomaly. METHODS: In the present study, a clinical and genetic investigation was performed in a consanguineous family of Pakistani origin, showing characteristic features of JS. Sanger sequencing was successfully used to identify the causative variant in CNNM4. Molecular dynamics (MD) simulations were performed to study the effect of amino acid change over CNNM4 protein. RESULTS: Sequence analysis of CNNM4 revealed a novel missense variant (c.1220G>T, p.Arg407Leu) in exon-1 encoding cystathionine-ß-synthase (CBS) domain. To comprehend the mutational consequences in the structure, the mutant p.Arg407Leu was modeled together with a previously reported variant (c.1484C>T, p.Thr495Ile) in the same domain. Additionally, docking analysis deciphered the binding mode of the adenosine triphosphate (ATP) cofactor. Furthermore, 60ns MD simulations were carried out on wild type (p.Arg407/p.Thr495) and mutants (p.Arg407Leu/p.Thr495Ile) to understand the structural and energetic changes in protein structure and its dynamic behavior. An evident conformational shift of ATP in the binding site was observed in simulated mutants disrupting the native ATP-binding mode. CONCLUSION: The novel identified variant in CNNM4 is the first report from the Pakistani population. Overall, the study is valuable and may give a novel insight into metal transport in visual function and biomineralization.

Molecular and in silico analyses validates pathogenicity of homozygous mutations in the NPR2 gene underlying variable phenotypes of Acromesomelic dysplasia, type Maroteaux.

Homozygous and/or heterozygous loss of function mutations in the natriuretic peptide receptor B (NPR2) have been reported in causing acromesomelic dysplasia, type Maroteaux with variable clinical features and idiopathic short stature with nonspecific skeletal deformities. On the other hand, gain of function mutations in the same gene result in overgrowth disorder suggesting that NPR2 and its ligand, natriuretic peptide precursor C (CNP), are the key players of endochondral bone growth. However, the precise mechanism behind phenotypic variability of the NPR2 mutations is not fully understood so far. In the present study, three consanguineous families of Pakistani origin (A, B, C) with variable phenotypes of acromesomelic dysplasia, type Maroteaux were evaluated at clinical and molecular levels. Linkage analysis followed by Sanger sequencing of the NPR2 gene revealed three homozygous mutations including p.(Leu314 Arg), p.(Arg371*), and p.(Arg1032*) in family A, B and C, respectively. In silico structural and functional analyses substantiated that a novel missense mutation [p.(Leu314 Arg)] in family A allosterically affects binding of NPR2 homodimer to its ligand (CNP) which ultimately results in defective guanylate cyclase activity. A nonsense mutation [p.(Arg371*)] in family B entirely removed the transmembrane domain, protein kinase domain and guanylate cyclase domains of the NPR2 resulting in abolishing its guanylate cyclase activity. Another novel mutation [p.(Arg1032*)], found in family C, deteriorated the guanylate cyclase domain of the protein and probably plundered its guanylate cyclase activity. These results suggest that guanylate cyclase activity is the most critical function of the NPR2 and phenotypic severity of the NPR2 mutations is proportional to the reduction in its guanylate cyclase activity.

A novel mutation in homeobox DNA binding domain of HOXC13 gene underlies pure hair and nail ectodermal dysplasia (ECTD9) in a Pakistani family.

BACKGROUND: Pure hair and nail ectodermal dysplasia (PHNED) is a congenital disorder of hair abnormalities and nail dysplasia. Both autosomal recessive and dominant inheritance fashion of PHNED occurs. In literature, to date, five different forms of PHNED have been reported at molecular level, having three genes known and two loci with no gene yet. METHODS: In this study, a four generations consanguineous family of Pakistani origin with autosomal recessive PHNED was investigated. Affected members exhibited PHNED phenotypes with involvement of complete hair loss and nail dysplasia. To screen for mutation in the genes (HOXC13, KRT74, KRT85), its coding exons and exons-intron boundaries were sequenced. The 3D models of normal and mutated HOXC13 were predicted by using homology modeling. RESULTS: Through investigating the family to known loci, the family was mapped to ectodermal dysplasia 9 (ECTD9) loci with genetic address of 12q13.13. Mutation screening revealed a novel missense mutation (c.929A > C; p.Asn310Thr) in homeobox DNA binding domain of HOXC13 gene in affected members of the family. Due to mutation, loss of hydrogen bonding and difference in potential energy occurs, which may resulting in alteration of protein function. CONCLUSION: This is the first mutation reported in homeodomain, while 5th mutation reported in HOXC13 gene causing PHNED.

Hypomorphic MKS1 mutation in a Pakistani family with mild Joubert syndrome and atypical features: Expanding the phenotypic spectrum of MKS1-related ciliopathies.

Postaxial polydactyly (PAP) is one of the most common congenital malformations observed in the general population. However, it can also occur as part of a syndrome. Unbiased genetic screening techniques such as exome sequencing are highly appropriate methods to provide a molecular diagnosis in patients with polydactyly due to the large number of mutated genes associated with it. The present study describes a consanguineous family of Pakistani origin with PAP, speech impairment, hearing impairment of variable degree, and proportionate short stature with no prominent intellectual disability or ophthalmological abnormalities. One affected individual of the family was subjected to exome sequencing which resulted in the identification of four homozygous variants including an in-frame deletion (c.1115_1117delCCT; p.(Ser372del) in MKS1, which was later shown to be the only variant segregating with the phenotype. In silico predictions supported the potential pathogenicity of the identified mutation. Additional clinical tests and MRI features of a patient in the family showed a molar tooth sign, which is a hallmark of Joubert syndrome. In conclusion, we have described a pathogenic variant in the MKS1 resulting in a mild Joubert syndrome phenotype, which broadens the spectrum of mutations in the MKS1. © 2016 Wiley Periodicals, Inc.

Genetics of human isolated acromesomelic dysplasia.

Acromesomelic dysplasia is a type of skeletal malformation affecting distal and middle segments of the extremities. It occurs in both isolated (non-syndromic) and syndromic forms. In later case, it shows association with cardiac, respiratory, neurological and genital abnormalities. Acromesomelic dysplasia segregates in autosomal recessive mode. Mutations in three genes (GDF5, NPR2, BMPR1B) have been reported to cause different forms of acromesomelic dysplasia. In the present review, we have discussed clinical spectrum, genetics and signalopathies of isolated acromesomelic dysplasias.

Disease causing homozygous variants in the human hairless gene.

BACKGROUND: Atrichia with papular lesions (APL) is a rare irreversible form of complete hair loss inherited in autosomal recessive manner. Hair loss is often followed by the appearance of multiple keratin-filled cysts or papules on exterior parts of the body. This phenotype results due to mutations in the human hairless gene (HR) mapped on chromosome 8p21.3. The present study was aimed to search for disease-causing sequence variants in the HR gene in five consanguineous families exhibiting features of APL. METHODS: Linkage in five Pakistani lineal consanguineous families, displaying features of APL, was tested using microsatellite markers flanking the HR gene on chromosome 8p21.3. After constructing the haplotypes, variants in the gene HR were searched by dideoxy-chain termination sequencing. RESULTS: Haplotype analysis established linkage in all five families to the HR gene located on chromosome 8p.21.3. Subsequently, sequencing HR identified a novel homozygous nonsense variant (c.2541G>A, p.Trp847*) in one and previously reported two pathogenic variants (p.Cys690*, p.Pro1157Arg) in the other four families. CONCLUSION: Mutations identified extend the spectrum of mutations in the HR gene resulting in APL. Characterizing the clinical spectrum resulting from the disease-causing homozygous variants in the HR gene will direct clinical care of the family members.

Homozygous sequence variants in the NPR2 gene underlying Acromesomelic dysplasia Maroteaux type (AMDM) in consanguineous families.

Acromesomelic dysplasia Maroteaux type (AMDM) is an autosomal recessive skeletal disorder characterized by disproportionate short stature with shortening of the acromesomelic sections of the limbs. AMDM is caused by mutations in the NPR2 gene located on chromosome 9p21-p12. The gene encodes the natriuretic peptide receptor B (NPR-B) that acts as an endogenous receptor for C-type natriuretic peptide (CNP). Both CNP and NPR-B are considered as important regulators of longitudinal growth. The study presented here investigated three consanguineous families (A, B, C) segregating AMDM in an autosomal recessive manner. Linkage in the families was established to the NPR2 gene on chromosome 9p12-21. Sequence analysis of the gene revealed two novel missense variants (p.Arg601Ser; p.Arg749Trp) in two families and a previously reported splice site variant (c.2986+2T>G) in the third family.

Novel homozygous mutations in the WNT10B gene underlying autosomal recessive split hand/foot malformation in three consanguineous families.

Split-hand/split-foot malformation (SHFM), representing variable degree of median clefts of hands and feet, is a genetically heterogeneous group of limb malformations with seven loci mapped on different human chromosomes. However, only 3 genes (TP63, WNT10B, DLX5) for the seven loci have been identified. The study, presented here, described three consanguineous Pakistani families segregating SHFM in autosomal recessive manner. Linkage in the families was searched by genotyping microsatellite markers and mutation screening of candidate gene was performed by Sanger DNA sequencing. Clinical features of affected members of these families exhibited SHFM phenotype with involvement of hands and feet. Genotyping using microsatellite markers mapped the families to WNT10B gene at SHFM6 on chromosome 12q13.11-q13. Subsequently, sequence analysis of WNT10B gene revealed a novel 4-bp deletion mutation (c.1165_1168delAAGT) in one family and 7-bp duplication (c.300_306dupAGGGCGG) in two other families. Structure-based analysis showed a significant conformational shift in the active binding site of mutated WNT10B (p.Lys388Glufs*36), influencing binding with Fzd8. The mutations identified in the WNT10B gene extend the body of evidence implicating it in the pathogenesis of SHFM.

Novel homozygous mutations in the genes ARL6 and BBS10 underlying Bardet-Biedl syndrome.

Bardet-Biedl syndrome (BBS) is an autosomal recessive disorder resulting from structural and functional defects in numerous organs. Frequent manifestations reported in the syndrome include obesity, renal dysplasia, cognitive impairment, postaxial polydactyly, pigmentary retinal degeneration and hypogonadism. To date, 17 genes causing BBS have been identified. Two of these BBS1 and BBS10 are the most frequently mutated genes. The present report describes two consanguineous families (A, B) with clinical manifestations of BBS. Linkage in the family A was established to ARL6 on chromosome 3q11.2, while family B showed linkage to BBS10 on chromosome 12q21.2. Sequence analysis revealed a novel homozygous missense mutation (c.281T>C, p.Ile94Thr) in the gene ARL6 in family A and a nonsense mutation (c.1075C>T, p.Gln359*) in the gene BBS10 in family B. Mutations identified in the present study extend the body of evidence implicating the genes ARL6 and BBS10 in causing Bardet-Biedl syndrome.

Novel mutations in natriuretic peptide receptor-2 gene underlie acromesomelic dysplasia, type maroteaux.

BACKGROUND: Natriuretic peptides (NPs) are peptide hormones that exert their biological actions by binding to three types of cell surface natriuretic peptide receptors (NPRs). The receptor NPR-B binding C-type natriuretic peptide (CNP) acts locally as a paracrine and/or autocrine regulator in a wide variety of tissues. Mutations in the gene NPR2 have been shown to cause acromesomelic dysplasia-type Maroteaux (AMDM), an autosomal recessive skeletal disproportionate dwarfism disorder in humans. METHODS: In the study, presented here, genotyping of six consanguineous families of Pakistani origin with AMDM was carried out using polymorphic microsatellite markers, which are closely linked to the gene NPR2 on chromosome 9p21-p12. To screen for mutations in the gene NPR2, all of its coding exons and splice junction sites were PCR amplified from genomic DNA of affected and unaffected individuals of the families and sequenced. RESULTS: Sequence analysis of the gene NPR2 identified a novel missence mutation (p.T907M) in five families, and a splice donor site mutation c.2986 + 2 T > G in the other family. CONCLUSION: We have described two novel mutations in the gene NPR2. The presence of the same mutation (p.T907M) and haplotype in five families (A, B, C, D, E) is suggestive of a founder effect.

A Novel ESRRB Deletion Is a Rare Cause of Autosomal Recessive Nonsyndromic Hearing Impairment among Pakistani Families.

Mutations in the estrogen-related receptor beta (ESRRB) gene is the underlying cause of autosomal recessive nonsyndromic hearing impairment (ARNSHI) due to the DFNB35 locus which maps to 14q24.3. A genome scan of a large consanguineous Pakistani pedigree with ARNSHI established linkage with a maximum multipoint LOD score of 4.2 to the 14q24 region and the region of homozygosity contained the ESRRB gene. Sequencing of the ESRRB gene using DNA samples from hearing-impaired family members uncovered a novel three-nucleotide deletion c.1018_1020delGAG (p.Glu340del). The deletion segregates with hearing impairment in the pedigree and was not observed in 500 control chromosomes. The deletion of glutamic acid residue occurs in the ligand-binding domain of ESRRB protein. It is expected that the deletion affects the ligand-binding activity of the domain in ESRRB, which leads to the ARNSHI.