Consanguineous Pakistani Family Research Articles

Next-generation sequencing to genetically diagnose a diverse range of inherited eye disorders in 15 consanguineous families from Pakistan

Inherited retinal dystrophies (IRDs) are characterized by photoreceptor dysfunction or degeneration. Clinical and phenotypic overlap between IRDs makes the genetic diagnosis very challenging and comprehensive genomic approaches for accurate diagnosis are frequently required. While there are previous studies on IRDs in Pakistan, causative genes and variants are still unknown for a significant portion of patients. Therefore, there is a need to expand the knowledge of the genetic spectrum of IRDs in Pakistan. Here, we recruited 52 affected and 53 normal individuals from 15 consanguineous Pakistani families presenting non-syndromic and syndromic forms of IRDs. We employed single molecule Molecular Inversion Probes (smMIPs) based panel sequencing and whole genome sequencing to identify the probable disease-causing variants in these families. Using this approach, we obtained a 93% genetic solve rate and identified 16 (likely) causative variants in 14 families, of which seven novel variants were identified in ATOH7, COL18A1, MERTK, NDP, PROM1, PRPF8 and USH2A while nine recurrent variants were identified in CNGA3, CNGB1, HGSNAT, NMNAT1, SIX6 and TULP1. The novel MERTK variant and one recurrent TULP1 variant explained the intra-familial locus heterogeneity in one of the screened families while two recurrent CNGA3 variants explained compound heterozygosity in another family. The identification of variants in known disease-associated genes emphasizes the utilization of time and cost-effective screening approaches for rapid diagnosis. The timely genetic diagnosis will not only identify any associated systemic issues in case of syndromic IRDs, but will also aid in the acceleration of personalized medicine for patients affected with IRDs.

Clinical genomics expands the link between erroneous cell division, primary microcephaly and intellectual disability.

Primary microcephaly is a rare neurogenic and genetically heterogeneous disorder characterized by significant brain size reduction that results in numerous neurodevelopmental disorders (NDD) problems, including mild to severe intellectual disability (ID), global developmental delay (GDD), seizures and other congenital malformations. This disorder can arise from a mutation in genes involved in various biological pathways, including those within the brain. We characterized a recessive neurological disorder observed in nine young adults from five independent consanguineous Pakistani families. The disorder is characterized by microcephaly, ID, developmental delay (DD), early-onset epilepsy, recurrent infection, hearing loss, growth retardation, skeletal and limb defects. Through exome sequencing, we identified novel homozygous variants in five genes that were previously associated with brain diseases, namely CENPJ (NM_018451.5: c.1856A > G; p.Lys619Arg), STIL (NM_001048166.1: c.1235C > A; p.(Pro412Gln), CDK5RAP2 (NM_018249.6 c.3935T > G; p.Leu1312Trp), RBBP8 (NM_203291.2 c.1843C > T; p.Gln615*) andCEP135(NM_025009.5 c.1469A > G; p.Glu490Gly). These variants were validated by Sanger sequencing across all family members, and in silico structural analysis. Protein 3D homology modeling of wild-type and mutated proteins revealed substantial changes in the structure, suggesting a potential impact on function. Importantly, all identified genes play crucial roles in maintaining genomic integrity during cell division, with CENPJ, STIL, CDK5RAP2, and CEP135 being involved in centrosomal function. Collectively, our findings underscore the link between erroneous cell division, particularly centrosomal function, primary microcephaly and ID.

Whole exome sequencing identified a homozygous novel variant in DOP1A gene in the Pakistan family with neurodevelopmental disabilities: case report and literature review.

Hereditary neurodevelopmental disorders (NDDs) are prevalent in poorly prognostic pediatric diseases, but the pathogenesis of NDDs is still unclear. Irregular myelination could be one of the possible causes of NDDs. Here, whole exome sequencing was carried out for a consanguineous Pakistani family with NDDs to identify disease-associated variants. The co-segregation of candidate variants in the family was validated using Sanger sequencing. The potential impact of the gene on NDDs has been supported by conservation analysis, protein prediction, and expression analysis. A novel homozygous variant DOP1A(NM_001385863.1):c.2561A>G was identified. It was concluded that the missense variant might affect the protein-protein binding sites of the critical MEC interaction region of DOP1A, and DOP1A-MON2 may cause stability deficits in Golgi-endosome protein traffic. Proteolipid protein (PLP) and myelin-associate glycoprotein (MAG) could be targets of the DOP1A-MON2 Golgi-endosome traffic complex, especially during the fetal stage and the early developmental stages. This further supports the perspective that disorganized myelinogenesis due to congenital DOP1A deficiency might cause neurodevelopmental disorders (NDDs). Our case study revealed the potential pathway of myelinogenesis-relevant NDDs and identified DOP1A as a potential NDDs-relevant gene in humans.

Deciphering the Genomic Complexity of Acromesomelic Dysplasia Type Maroteaux: Insights from a Consanguineous Pakistani Family

Purpose: This study delves into the genetic basis of Acromesomelic Dysplasia (AMD), a rare skeletal disorder, particularly focusing on the Maroteaux type within a consanguineous Pakistani family. The purpose is to explore the genetic landscape beyond known mutations and understand the underlying causes of AMD.
 Methodology: A comprehensive approach was adopted, combining detailed clinical examinations with advanced genomic methodologies. Ethical clearance was obtained, and pedigree design facilitated the identification of affected individuals. Molecular techniques including DNA extraction and linkage analysis were conducted to investigate known AMD-related genes such as NPR2, BMPR1B, GDF5, GALNS, GLB1, and GHR. Whole-genome sequencing was emphasized despite financial constraints to uncover potential novel genetic loci associated with AMD.
 Findings: Despite exhaustive analysis, no mutations in known AMD-related genes were identified within the studied family. Linkage analysis did not correlate with any known genetic loci, suggesting the presence of unidentified genetic elements contributing to AMD. Autosomal recessive inheritance was confirmed through pedigree and molecular scrutiny, highlighting the complexity of AMD genetics.
 Unique Contribution to Theory, Policy and Practice: This research underscores the importance of employing advanced genomic strategies, such as whole-genome sequencing, in decoding rare genetic disorders like AMD. By revealing the limitations of current diagnostic approaches and advocating for collaborative efforts and resource pooling, this study contributes to the advancement of genetic counseling, therapeutic interventions, and precision medicine in rare genetic disorders.

A Novel Missense Variant in the <i>CHST3</i> Underlies Spondyloepiphyseal Dysplasia with Congenital Joint Dislocations

<b><i>Background:</i></b> Spondyloepiphyseal dysplasia (SED) is characterized by skeletal dysplasia and multiple joint dislocations. SEDs encompass various types, such as SED congenita, SED tarda (SED-T), SED with congenital joint dislocations (SED-CJD), SED stanescu, and SED-T with progressive arthropathy. <b><i>Methods and Results:</i></b> In the present study, we clinically and genetically characterized a consanguineous Pakistani family with SED-CJD. The affected member showed large joint dislocation, spinal deformities, and previously unreported facial features. Exome sequencing followed by Sanger sequencing revealed a missense variant, [c.601T>A; p.(Tyr201Asn)], in the <i>CHST3</i>. <b><i>Conclusion:</i></b> This study has not only expended the mutation spectrum in the gene <i>CHST3</i> but also will facilitate diagnosis and genetic counseling of related features in the Pakistani population.

Multi-gene panel sequencing in highly consanguineous families and patients with congenital forms of skeletal dysplasias.

Skeletal dysplasias (SKDs) are a heterogeneous group of more than 750 genetic disorders characterized by abnormal development, growth, and maintenance of bones or cartilage in the human skeleton. SKDs are often caused by variants in early patterning genes and in many cases part of multiple malformation syndromes and occur in combination with non-skeletal phenotypes. The aim of this study was to investigate the underlying genetic cause of congenital SKDs in highly consanguineous Pakistani families, as well as in sporadic and familial SKD cases from India using multigene panel sequencing analysis. Therefore, we performed panel sequencing of 386 bone-related genes in 7 highly consanguineous families from Pakistan and 27 cases from India affected with SKDs. In the highly consanguineous families, we were able to identify the underlying genetic cause in five out of seven families, resulting in a diagnostic yield of 71%. Whereas, in the sporadic and familial SKD cases, we identified 12 causative variants, corresponding to a diagnostic yield of 44%. The genetic heterogeneity in our cohorts was very high and we were able to detect various types of variants, including missense, nonsense, and frameshift variants, across multiple genes known to cause different types of SKDs. In conclusion, panel sequencing proved to be a highly effective way to decipher the genetic basis of SKDs in highly consanguineous families as well as sporadic and or familial cases from South Asia. Furthermore, our findings expand the allelic spectrum of skeletal dysplasias.

Mutational analysis in different genes underlying severe combined immunodeficiency in seven consanguineous Pakistani families.

Severe Combined Immunodeficiency (SCID) is an autosomal recessive inborn error of immunity (IEI) characterized by recurrent chest and gastrointestinal (GI) infections and in some cases associated with life-threatening disorders. This current study aims to unwind the molecular etiology of SCID and also extended the patients' phenotype associated withidentified particular variants. Herein, we present 06 disease-causing variants identified in 07 SCID-patients in three different SCID related genes. Whole Exome Sequencing (WES) followed by Sanger Sequencing was employed to explore genetic variations. The results included identification of two previously reported heterozygous variants in homozygous form for the first time in RAG1gene [(p.Arg410Gln);(p.Arg737His)], followed by a recurrent variant (p.Trp959*) in RAG1, a novel variant in IL2RG (p.Asp48Lfs*24), a recurrent variant in IL2RG (p.Gly271Glu) and a recurrent variant in DCLRE1C (p.Arg191*) gene. To conclude, the immune-profiling and WES revealed two novel, two as homozygous state for the first time, and two recurrent disease causing variants contributing valuably to our existing knowledge of SCID.

Mutational analysis of consanguineous families and their targeted therapy against dwarfism

Dwarfism is a medical term used to describe individuals with a height-vertex measurement that falls below two standard deviations (−2SD) or the third percentile for their gender and age. Normal development of growth is a complicated dynamic procedure that depends upon the coordination of different aspects involving diet, genetics, and biological aspects like hormones in equilibrium. Any severe or acute pathologic procedure may disturb the individual’s normal rate of growth. In this research, we examined four (A–D) Pakistani consanguineous families that exhibited syndromic dwarfism, which was inherited in an autosomal recessive pattern. The genomic DNA of each family member was extracted by using phenol-chloroform and Kit methods. Whole Exome Sequencing (WES) of affected family members (IV-11, III-5, IV-4 and III-13) from each group was performed at the Department of Medical Genetics, University of Antwerp, Belgium. After filtering the exome data, the mutations in PPM1F, FGFR3, ERCC2, and PCNT genes were determined by Sanger sequencing of each gene by using specific primers. Afterward, FGFR3 was found to be a suitable drug target among all the mutations to treat achondroplasia also known as disproportionate dwarfism. BioSolveIT softwares were used to discover the lead active inhibitory molecule against FGFR3. This research will not only provide short knowledge to the concerned pediatricians, researchers, and family physicians for the preliminary assessment and management of the disorder but also provide a lead inhibitor for the treatment of disproportionate dwarfism. Communicated by Ramaswamy H. Sarma

Investigating the effects of a single ASPM variant (c.8508_8509) on brain architecture among siblings in a consanguineous Pakistani family.

Autosomal Recessive Primary Microcephaly (MCPH) is a rare, neurodevelopmental disorder associated with mild to severe mental retardation. It is characterized by reduced cerebral cortex that ultimately leads to reduction in skull size less than - 3S.D below the mean for normal individuals having same age and sex. Till date, 30 known loci have been reported for MCPH. In the present study, Sanger sequencing was performed followed by linkage analysis to validate the mutation in ASPM gene of the consanguineous Pakistani clans. Bioinformatics tools were also used to confirm the pathogenicity of the diseased variant in the gene. MRI scan was used to compare the brain structure of both the affected individuals (Aslam et al. in Kinnaird's 2nd International Conference on Science, Technology and Innovation, Lahore, 2023). Our study described a consanguineous family with two patients with a known ASPM (MCPH5) variant c.8508_8509delGA causing a frameshift mutation in exon 18 which located in calmodulin-binding IQ domain of the ASPM protein. The salient feature of this study is that a single variant led to significantly distinct changes in the architecture of brain of both siblings which is further confirmed by MRI results. The computation analysis showed that the change in the conservation of this residue cause this variant highly pathogenic. Carrier screening and genetic counselling were also remarkable features of this study (Aslam et al. in Kinnaird's 2nd International Conference on Science, Technology and Innovation, Lahore, 2023). This study explores the extraordinary influence of a single ASPM variant on divergent brain structure in consanguineous siblings and enable us to reduce the incidence of further microcephalic cases in this Pakistani family (Aslam et al. in Kinnaird's 2nd International Conference on Science, Technology and Innovation, Lahore, 2023).

Macrocephaly and Digital Anomalies Expand the Phenotypic Spectrum of PGAP2 Variants in Hyperphosphatasia with Impaired Intellectual Development Syndrome 3 (HPMRS3)

Glycosylphosphatidylinositols (GPIs) anchor over 150 proteins as GPI-anchored proteins (GPI-APs) with crucial roles in diverse biological processes. The highly conserved biosynthesis of GPI-APs involves precise steps with at least 21 genes, categorized as PIG and PGAP genes. Pathogenic variants in these genes are linked to human diseases, highlighting the importance of each biosynthesis step. PGAP2 stands out among these genes due to its association with an expanded clinical spectrum of neurodevelopmental disorder (NDD) phenotypes with biallelic pathogenic variants. We present four patients from two families, one consanguineous and the other nonconsanguineous, each displaying distinct clinical presentations, including intellectual disability, hyperphosphatasia, hearing impairment, and epilepsy, as well as craniofacial and digital anomalies. Genetic analyses revealed homozygous and novel compound heterozygous missense variants in PGAP2 in four affected individuals, confirming the molecular diagnosis of hyperphosphatasia with impaired intellectual development syndrome 3 (HPMRS3). Importantly, the three amino acids affected by missense variants exhibit complete conservation in 10 vertebrate species, illuminating their crucial role in the gene’s functionality. Protein modeling provided additional evidence for the pathogenicity of the three substitutions, demonstrating their detrimental impact on protein folding and putative protein-protein interactions, ultimately leading to impaired protein function. The four patients in our study displayed common phenotypic features, such as brachydactyly, camptodactyly, and syndactyly, which have not been previously documented in individuals with PGAP2 variants. Notably, the occurrence of macrocephaly in two affected brothers from a consanguineous Pakistani family represents a novel finding. These previously unreported digital anomalies, along with macrocephaly and the identification of novel compound heterozygous variants, contribute to the expansion of the phenotypic and genotypic spectrum of HPMRS3 associated with PGAP2 variants.

Exome sequencing identifies homozygous variants in MBOAT7 associated with neurodevelopmental disorder.

Intellectual disability (ID) is a large group of neurodevelopmental disorders characterized by a congenital limitation in intellectual functioning (reasoning, learning, and problem solving), adaptive behavior (conceptual, social, and practical skills), originated at birth and manifested before the age of 18. By whole exome sequencing of five consanguineous Pakistani families presenting hallmark features of ID, global developmental delay, aggressive and self-injurious behaviors, microcephaly, febrile seizures and facial dysmorphic features, we identified three novel homozygous missense variants (NM_024298.5: c.588G > T; p.Trp196Cys, c.736 T > C; p.Tyr246His and c.524A > C; p. Asp175Ala) and one rare homozygous in-frame deletion variant (c.758_778del;p.Glu253_Ala259del) in membrane-bound O-acyltransferase family member 7 (MBOAT7) gene previously associated with autosomal recessive neurodevelopmental disorder. The segregation of the variants was validated by Sanger sequencing in all family members. In silico homology modeling of wild-type and mutated proteins revealed substantial changes in the structure of both proteins, indicating a possible effect on function. The identification and validation of new pathogenic MBOAT7 variants in five cases of autosomal recessive ID further highlight the importance of this genes in proper brain function and development.

Sequence variants in different genes underlying Bardet-Biedl syndrome in four consanguineous families.

Bardet-Biedl Syndrome (BBS) is a rare (1:13,500-1-160,000) heterogeneous congenital disorder, characterized by postaxial polydactyly, obesity, hypogonadism, rod-cone dystrophy, cognitive impairment, and renal abnormalities (renal cystic dysplasia, anatomical malformation). To date about twenty-five genes have been identified to cause BBS, which accounts for about 80% of BBS diagnosis. In the current study, we have performed mutational screening of four Pakistani consanguineous families (A-D) with clinical manifestation of BBS by microsatellite-based genotyping and whole exome sequencing. Analysis of the data revealed four variants, including a novel/unique inheritance pattern of compound heterozygous variants, p.(Ser40*) and p.(Thr259Leufs*21), in MKKS gene, novel homozygous variant, p.(Gly251Val)] in BBS7 gene and two previously reported p.(Thr259Leufs*21) in MKKS and p.(Met1Lys) in BBS5 gene. The variants were found segregated with the disorder within the families. The study not only expanded mutations spectrum in the BBS genes, but this will facilitate diagnosis and genetic counselling of families carrying BBS related phenotypes in Pakistani population.

Truncated DNM1 variant underlines developmental delay and epileptic encephalopathy.

Developmental and epileptic encephalopathies (DEEs) signify a group of heterogeneous neurodevelopmental disorder associated with early-onset seizures accompanied by developmental delay, hypotonia, mild to severe intellectual disability, and developmental regression. Variants in the DNM1 gene have been associated with autosomal dominant DEE type 31A and autosomal recessive DEE type 31B. In the current study, a consanguineous Pakistani family consisting of a proband (IV-2) was clinically evaluated and genetically analyzed manifesting in severe neurodevelopmental phenotypes. WES followed by Sanger sequencing was performed to identify the disease-causing variant. Furthermore, 3D protein modeling and dynamic simulation of wild-type and mutant proteins along with reverse transcriptase (RT)-based mRNA expression were checked using standard methods. Data analysis of WES revealed a novel homozygous non-sense variant (c.1402G>T; p. Glu468*) in exon 11 of the DNM1 gene that was predicted as pathogenic class I. Variants in the DNM1 gene have been associated with DEE types 31A and B. Different bioinformatics prediction tools and American College of Medical Genetics guidelines were used to verify the identified variant. Sanger sequencing was used to validate the disease-causing variant. Our approach validated the pathogenesis of the variant as a cause of heterogeneous neurodevelopmental disorders. In addition, 3D protein modeling showed that the mutant protein would lose most of the amino acids and might not perform the proper function if the surveillance non-sense-mediated decay mechanism was skipped. Molecular dynamics analysis showed varied trajectories of wild-type and mutant DNM1 proteins in terms of root mean square deviation, root mean square fluctuation and radius of gyration. Similarly, RT-qPCR revealed a substantial reduction of the DNM1 gene in the index patient. Our finding further confirms the association of homozygous, loss-of-function variants in DNM1 associated with DEE type 31B. The study expands the genotypic and phenotypic spectrum of pathogenic DNM1 variants related to DNM1-associated pathogenesis.

Genome Sequencing of Consanguineous Family Implicates Ubiquitin-Specific Protease 53 (USP53) Variant in Psychosis/Schizophrenia: Wild-Type Expression in Murine Hippocampal CA 1-3 and Granular Dentate with AMPA Synapse Interactions.

Psychosis is a severe mental disorder characterized by abnormal thoughts and perceptions (e.g., hallucinations) occurring quintessentially in schizophrenia and in several other neuropsychiatric disorders. Schizophrenia is widely considered as a neurodevelopmental disorder that onsets during teenage/early adulthood. A multiplex consanguineous Pakistani family was afflicted with severe psychosis and apparent autosomal recessive transmission. The first-cousin parents and five children were healthy, whereas two teenage daughters were severely affected. Structured interviews confirmed the diagnosis of DSM-V schizophrenia. Probands and father underwent next-generation sequencing. All available relatives were subjected to confirmatory Sanger sequencing. Homozygosity mapping and directed a priori filtering identified only one rare variant [MAF < 5(10)-5] at a residue conserved across vertebrates. The variant was a non-catalytic deubiquitinase, USP53 (p.Cys228Arg), predicted in silico as damaging. Genome sequencing did not identify any other potentially pathogenic single nucleotide variant or structural variant. Since the literature on USP53 lacked relevance to mental illness or CNS expression, studies were conducted which revealed USP53 localization in regions of the hippocampus (CA 1-3) and granular dentate. The staining pattern was like that seen with GRIA2/GluA2 and GRIP2 antibodies. All three proteins coimmunoprecipitated. These findings support the glutamate hypothesis of schizophrenia as part of the AMPA-R interactome. If confirmed, USP53 appears to be one of the few Mendelian variants potentially causal to a common-appearing mental disorder that is a rare genetic form of schizophrenia.

Homozygous Mutations in GDAP1 and MFN2 Genes Resulted in Autosomal Recessive Forms of Charcot-Marie-Tooth Disease in Consanguineous Pakistani Families.

Charcot-Marie-Tooth disease (CMT) is a heritable neurodegenerative disease of peripheral nervous system diseases in which more than 100 genes and their mutations are associated. Two consanguineous families Dera Ghazi Khan (PAK-CMT1-DG KHAN) and Layyah (PAK-CMT2-LAYYAH) with multiple CMT-affected subjects were enrolled from Punjab province in Pakistan. Basic epidemiological data were collected for the subjects. Nerve conduction study (NCS) and electromyography (EMG) were performed for the patients. Whole-exome sequencing (WES) followed by Sanger sequencing was applied to report the genetic basic of CMT. The NCS findings revealed that sensory and motor nerve conduction velocities for both families were <38 m/s. EMG presented denervation, neuropathic motor unit potential, and reduced interference pattern of peripheral nerves. WES identified that a novel nonsense mutation (c. 226 G>T) in GADP1 gene and a previously known missense mutation in MFN2 gene (c. 334 G>A) cause CMT4A (Charcot-Marie-Tooth disease type 4A) in the PAK-CMT1-DG KHAN family and CMT2A (Charcot-Marie-Tooth disease type 2A) in the PAK-CMT2-LAYYAH family, respectively. Mutations followed Mendelian pattern with autosomal recessive mode of inheritance. Multiple sequence alignment by Clustal Omega indicated that mutation-containing domain in both genes is highly conserved, and in situ analysis revealed that both mutations are likely to be pathogenic. We reported that a novel nonsense mutation and a previously known missense mutation in GAPD1 gene and MFN2 gene, respectively, cause CMT in consanguineous Pakistani families.

Identification of novel homozygous variants in FOXE3 and AP4M1 underlying congenital syndromic anophthalmia and microphthalmia.

Anophthalmia and microphthalmia are severe developmental ocular disorders that affect the size of the ocular globe and can be unilateral or bilateral. The disease is found in syndromic as well as non-syndromic forms. It is genetically caused by chromosomal aberrations, copy number variations and single gene mutations, along with non-genetic factors such as viral infections, deficiency of vitamin A and an exposure to alcohol or drugs during pregnancy. To date, more than 30 genes having different modes of inheritance patterns are identified as causing anophthalmia and microphthalmia. In the present study, a clinical and genetic analysis was performed of six patients with anophthalmia and microphthalmia and/or additional phenotypes of intellectual disability, developmental delay and cerebral palsy from a large consanguineous Pakistani family. Whole exome sequencing followed by data analysis for variants prioritization and validation through Sanger sequencing was performed to identify the disease causing variant(s). American College of Medical Genetics and Genomics (ACMG) guidelines were applied to classify clinical interpretation of the prioritized variants. Clinical investigations revealed that the affected individuals are afflicted with anophthalmia. Three of the patients showed additional phenotype of intellectual disability, developmental delays and other neurological symptoms. Whole exome sequencing of the DNA samples of the affected members in the family identified a novel homozygous stop gain mutation (NM_012186: c.106G>T: p.Glu36*) in Forkhead Box E3 (FOXE3) gene shared by all affected individuals. Moreover, patients segregating additional phenotypes of spastic paraplegia, intellectual disability, hearing loss and microcephaly showed an additional homozygous sequence variant (NM_004722: c.953G>A: p.Arg318Gln) in AP4M1. Sanger sequencing validated the correct segregation of the identified variants in the affected family. ACMG guidelines predicted the variants to be pathogenic. We have investigated first case of syndromic anophthalmia caused by variants in the FOXE3 and AP4M1. The present findings are helpful for understanding pathological role of the mutations of the genes in syndromic forms of anophthalmia. Furthermore, the study signifies searching for the identification of second variant in families with patients exhibiting variable phenotypes. In addition, the findings will help clinical geneticists, genetic counselors and the affected family with respect to prenatal testing, family planning and genetic counseling.

Variants in EFCAB7 underlie nonsyndromic postaxial polydactyly.

Polydactyly is the most common limb malformation that occurs in 1.6-10.6 per one thousand live births, with incidence varying with ancestry. The underlying gene has been identified for many of the ~100 syndromes that include polydactyly. While for the more common form, nonsydromic polydactyly, eleven candidate genes have been reported. We investigated the underlying genetic cause of autosomal recessive nonsyndromic postaxial polydactyly in four consanguineous Pakistani families. Some family members with postaxial polydactyly also present with syndactyly, camptodactyly, or clinodactyly. Analysis of the exome sequence data revealed two novel homozygous frameshift deletions in EFCAB7: [c.830delG;p.(Gly277Valfs*5)]; in three families and [c.1350_1351delGA;p.(Asn451Phefs*2)] in one family. Sanger sequencing confirmed that these variants segregated with postaxial polydactyly, i.e., family members with postaxial polydactyly were found to be homozygous while unaffected members were heterozygous or wild type. EFCAB7 displays expressions in the skeletal muscle and on the cellular level in cilia. IQCE-EFCAB7 and EVC-EVC2 are part of the heterotetramer EvC complex, which is a positive regulator of the Hedgehog (Hh) pathway, that plays a key role in limb formation. Depletion of either EFCAB7 or IQCE inhibits induction of Gli1, a direct Hh target gene. Variants in IQCE and GLI1 have been shown to cause nonsyndromic postaxial polydactyly, while variants in EVC and EVC2 underlie Ellis van Creveld and Weyers syndromes, which include postaxial polydactyly as a phenotype. This is the first report of the involvement of EFCAB7 in human disease etiology.

Whole exome sequencing reveals a missense mutation in the ADA gene causing severe combined immune deficiency in a Pakistani family ADA gene mutation causing SCID

BACKGROUND &amp; OBJECTIVE: Human adenosine deaminase deficiency, OMIM 102700 (ADA deficiency) is a genetic disorder which causes severe combined immunodeficiency (SCID). The enzyme adenosine deaminase is a housekeeping in nature and is involved in purine catabolism by catalyzing irreversible deamination of adenosine and deoxyadenosine. The SCID patients’ immune system is unable to fight off most of the bacterial and fungal infections due to profound lymphopenia (T-B-NK+). About 20% of the SCID patients are genetically homozygous for defective ADA gene. In our study we aimed to find out genetic variant in ADA gene in a family carrying severe combined immune deficiency. Objective in the current study is to unravel and characterize the molecular cause of the patient suffering from by birth SCID.&#x0D; METHODOLOGY: In the present study we enrolled a 6-month-old female SCID patient belonging to highly consanguineous Pakistani family. Patient clinical features included repeated chest infection with failure to thrive, fever and chronic diarrhea. Whole blood samples from patient, parents and healthy siblings were acquired in EDTA tubes. DNA was extracted from all the blood samples.&#x0D; RESULTS: Flowcytometry revealed lymphopenia (T-B-NK+) type of SCID. Whole Exome Sequencing (WES) identified a one nucleotide change (c.716G&gt;A) in ADA gene exon 8. The segregation of the identified variant in the family was confirmed through Sanger Sequencing.&#x0D; CONCLUSION: In this study, we presented detailed clinical and genetic description of patient suffering from severe combined immune deficiency. The immunological and genetic findings presented in this study will facilitate early diagnosis of the disease. Segregation of the identified variant in the family members will also aid in genetic counseling the family.

Loss-of-function variants in KCTD19 cause non-obstructive azoospermia in humans

Azoospermia is a significant cause of male infertility, with non-obstructive azoospermia (NOA) being the most severe type of spermatogenic failure. NOA is mostly caused by congenital factors, but our understanding of its genetic causes is very limited. Here, we identified a frameshift variant (c.201_202insAC, p.Tyr68Thrfs∗17) and two nonsense variants (c.1897C>T, p.Gln633∗; c.2005C>T, p.Gln669∗) in KCTD19 (potassium channel tetramerization domain containing 19) from two unrelated infertile Chinese men and a consanguineous Pakistani family with three infertile brothers. Testicular histological analyses revealed meiotic metaphase I (MMI) arrest in the affected individuals. Mice modeling KCTD19 variants recapitulated the same MMI arrest phenotype due to severe disrupted individualization of MMI chromosomes. Further analysis showed a complete loss of KCTD19 protein in both Kctd19 mutant mouse testes and affected individual testes. Collectively, our findings demonstrate the pathogenicity of the identified KCTD19 variants and highlight an essential role of KCTD19 in MMI chromosome individualization.

Structural and functional insights into a novel homozygous missense pathogenic variant in CUL7 identified in consanguineous Pakistani family

3M syndrome is a rare genetic familial disorder characterized by short stature, growth retardation, facial dysmorphism, skeletal abnormalities, fleshy protruding heels, and normal intelligence, caused by mutations in the CUL7, OBSL1 and CCDC8 genes. In the present study, a novel homozygous missense variant of CUL7 (NP_001161842.1, c.4493T > C, p.L1498P) has been identified in a consanguineous Pakistani family by whole exome sequencing. In silico structural evaluation, molecular docking and simulation studies of mutant CUL7 provides substantial evidence about its crucial role in the progression of discussed ailment. The newly discovered variant significantly altered the protein’s three dimensional structure, leading to abnormal interaction with binding proteins. This computational and experimental investigation provides useful information to drug developers for the synthesis of novel therapeutics against the discussed ailment. Communicated by Ramaswamy H. Sarma