Syntaxin 4 is essential for hearing in human and zebrafish.

Congenital hearing impairment (HI) is a genetically highly heterogeneous disorder in which prompt recognition and intervention are crucial to optimize outcomes. In this study, we used exome sequencing to investigate a large consanguineous Pakistani family with eight affected individuals showing bilateral severe-to-profound HI. This identified a homozygous splice region variant in STX4 (c.232 + 6T>C), which causes exon skipping and a frameshift, that segregated with HI (two-point logarithm of odds (LOD) score = 5.9). STX4, a member of the syntaxin family, is a component of the SNARE machinery involved in several vesicle transport and recycling pathways. In silico analysis showed that murine orthologue Stx4a is highly and widespread expressed in the developing and adult inner ear. Immunofluorescent imaging revealed localization of STX4A in the cell body, cell membrane and stereocilia of inner and outer hair cells. Furthermore, a morpholino-based knockdown of stx4 in zebrafish showed an abnormal startle response, morphological and developmental defects, and a disrupted mechanotransduction function in neuromast hair cells measured via FM1-43 uptake. Our findings indicate that STX4 dysfunction leads to HI in humans and zebrafish and supports the evolutionary conserved role of STX4 in inner ear development and hair cell functioning.

Clinical and genetic investigation of 14 families with various forms of short stature syndromes.

Skeletal dysplasias are a heterogeneous group of disorders presenting mild to lethal defects. Several factors, such as genetic, prenatal, and postnatal environmental may contribute to reduced growth. Fourteen families of Pakistani origin, presenting the syndromic form of short stature either in the autosomal recessive or autosomal dominant manner were clinically and genetically investigated to uncover the underlying genetic etiology. Homozygosity mapping, whole exome sequencing, and Sanger sequencing were used to search for the disease-causing gene variants. In total, we have identified 13 sequence variants in 10 different genes. The variants in the HSPG2 and XRCC4 genes were not reported previously in the Pakistani population. This study will expand the mutation spectrum of the identified genes and will help in improved diagnosis of the syndromic form of short stature in the local population.

Satisfaction of patients in government medical teaching institutes of Peshawar Khyber Pakhtunkhwa, Pakistan: a cross-sectional analytical study.

Objective: To evaluate patient satisfaction and its associated factors in teaching hospitals. METHODS: The cross-sectional, analytical study was conducted from September to December 2022 at three publicsector medical teaching hospitals in Peshawar, Pakistan, and comprised adult patients of either admitted to various hospital wards for at least 2 days. Data was collected using a predesigned a closed-ended questionnaire assessing patient satisfaction in different domains like, facilitation at the admission, professional knowledge and skills of the attending doctors, quality of diagnostic and nursing services, and basic amenities. Data was analysed using SPSS version origin Pro 2022a. RESULTS: There were 473 patients with a male-female ratio of 3:1, with mean age 43.3+14.7 years (range: 11-85 years), and mean hospital stay 5.96+3.37 days (range: 2-18 days). Of the 2,365 response statements for facilitation at the admission counter, 2,051(87%) were positive; of the 2,365 statements for attending doctors, 2,012(85%) were positive; of the 2,838 statements for nursing care, 2,122(75%) were positive; of 946 statements for diagnostic services, 627(66%) were positive; and of the 3,311 statements for basic amenities at the hospital, 1,246(38%) were positive. Overall, of the 11,825 response statements, 8058(68%) were positive. The patient satisfaction was significantly co-related with education and hospital stay (p<0.05). Conclusion: Patients were found to be generally satisfied with healthcare services, but not with the provision of basic amenities.

Nonsense variant in a consanguineous family expands the phenotype of KPTN gene-related syndrome to include hearing impairment.

A short report with two affected siblings from consanguineous family born with intellectual disability, motor disability, language deficit, and hearing impairment and found to carry biallelic nonsense variant in KPTN gene known to be associated with KPTN gene related syndrome.

A novel homozygous missense substitution p.Thr313Ile in the PDE6B gene underlies autosomal recessive retinitis pigmentosa in a consanguineous Pakistani family.

BACKGROUND: Retinitis pigmentosa (RP) is one of the most frequent hereditary retinal diseases that often starts with night blindness and eventually leads to legal blindness. Our study aimed to identify the underlying genetic cause of autosomal recessive retinitis pigmentosa (arRP) in a consanguineous Pakistani family. METHODS: Following a detailed ophthalmological examination of the patients by an ophthalmologist, whole-exome sequencing was performed on the proband's DNA to delineate the genetic cause of RP in the family. In-depth computational methods, in-silico analysis, and familial co-segregation study were performed for variant detection and validation. RESULTS: We studied an inbred Pakistani family with two siblings affected by retinitis pigmentosa. The proband, a 32 years old female, was clinically diagnosed with RP at the age of 6 years. A classical night blindness symptom was reported in the proband since her early childhood. OCT report showed a major reduction in the outer nuclear layer and the ellipsoid zone width, leading to the progression of the disease. Exome sequencing revealed a novel homozygous missense mutation (c.938C > T;p.Thr313Ile) in exon 12 of the PDE6B gene. The mutation p.Thr313Ile co-segregated with RP phenotype in the family. The altered residue (p.Thr313) was super conserved evolutionarily across different vertebrate species, and all available in silico tools classified the mutation as highly pathogenic. CONCLUSION: We present a novel homozygous pathogenic mutation in the PDE6B gene as the underlying cause of arRP in a consanguineous Pakistani family. Our findings highlight the importance of missense mutations in the PDE6B gene and expand the known mutational repertoire of PDE6B-related RP.

Novel splicing-site mutation in DCAF17 gene causing Woodhouse-Sakati syndrome in a large consanguineous family.

BACKGROUND: Woodhouse-Sakati syndrome is a rare autosomal recessive disease with endocrine and neuroectodermal aberrations with heterogeneous phenotypes and disease course. The most common phenotypes of the disease are progressive sensorineural hearing loss and alopecia, mild-to-moderate mental retardation and hypogonadism. The disease results from mutations in the DCAF17 gene. METHOD: Here, we reported a large consanguineous pedigree with multiple affected individuals with Woodhouse-Sakati syndrome phenotypes. Laboratory tests confirmed the endocrine perturbance in affected individuals. To find out the underlying genetic change, whole-exome sequencing was carried out. RESULT: Analysis of the exome data identified a splicing-site deletion NM_025000.3:c.1423-1_1425delGACA in DCAF17 gene. Sanger sequencing confirmed the co-segregation of the variant with the disease phenotypes in the family. CONCLUSION: The variant is predicted to cause aberrant splicing, i.e., exon skipping, resulting in the translation of a truncated functionless protein which results in appearance of typical phenotypic features and clinical laboratory findings of Woodhouse-Sakati syndrome in affected members of the family.

Cobalt Single Atom Incorporated in Ruthenium Oxide Sphere: A Robust Bifunctional Electrocatalyst for HER and OER.

The development of highly active and stable bifunctional noble-metal-based electrocatalysts for the hydrogen evolution reaction (HER) and the oxygen evolution reaction (OER) is a crucial goal for clean and renewable energy, which still remains challenging. Herein, we report an efficient and stable catalyst comprising a Co single atom incorporated in an RuO2 sphere for HER and OER, in which the Co single atom in the RuO2 sphere was confirmed by XAS, AC-STEM, and DFT. This tailoring strategy uses a Co single atom to modify the electronic structures of the surrounding Ru atoms and thereby remarkably elevates the electrocatalytic activities. The catalyst requires ultralow overpotentials, 45 mV for HER and 200 mV for OER, to deliver a current density of 10 mA cm-2 . The theoretical calculations reveal that the energy barriers for HER and OER are lowered after incorporation of a cobalt single atom.

BBS5 and INPP5E mutations associated with ciliopathy disorders in families from Pakistan.

Ciliopathies are a clinically and genetically heterogeneous group of disorders often exhibiting phenotypic overlap and caused by abnormalities in the structure or function of cellular cilia. As such, a precise molecular diagnosis is important for guiding clinical management and genetic counseling. In the present study, two Pakistani families comprising individuals with overlapping clinical features suggestive of a ciliopathy syndrome, including intellectual disability, obesity, congenital retinal dystrophy, and hypogonadism (in males), were investigated clinically and genetically. Whole-exome sequencing identified the likely causes of disease as a novel homozygous frameshift mutation (NM_152384.2: c.196delA; p.(Arg66Glufs*12); family 1) in BBS5, and a nonsense mutation (NM_019892.5:c.1879C>T; p.Gln627*; family 2) in INPP5E, previously reported in an extended Pakistani family with MORM syndrome. Our findings expand the molecular spectrum associated with BBS5 mutations in Pakistan and provide further supportive evidence that the INPP5E mutation is a common cause of ciliopathy in Northern Pakistan, likely representing a regional founder mutation. This study also highlights the value of genomic studies in Pakistan for families affected by rare heterogeneous developmental disorders and where clinical phenotyping may be limited by geographical and financial constraints. The identification of the spectrum and frequency of disease-causing variants within this setting enables the development of population-specific genetic testing strategies targeting variants common to the local population and improving health care outcomes.

Identification of CACNA1D variants associated with sinoatrial node dysfunction and deafness in additional Pakistani families reveals a clinical significance.

Sinoatrial node dysfunction and deafness (SANDD) syndrome is rare and characterized by a low heart beat and severe-to-profound deafness. Additional features include fatigue, dizziness, and episodic syncope. The sinoatrial node (SAN) drives heart automaticity and continuously regulates heart rate. The CACNA1D gene encoding the Cav1.3 protein expressed in inner hair cells, atria and SAN, induces loss-of-function in channel activity and underlies SANDD. To date, only one variant c.1208_1209insGGG:p.(G403_V404insG) has been reported for SANDD syndrome. We studied five Pakistani families with SANDD and characterized a new missense variant p.(A376V) in CACNA1D in one family, and further characterized the founder variant p.(G403_V404insG) in four additional pedigrees. We show that affected individuals in the four families which segregate p.(G403_V404insG) share a 1.03 MB haplotype on 3p21.1 suggesting they share a common distant ancestor. In conclusion, we identified new and known variants in CACNA1D in five Pakistani families with SANDD. This study is of clinical importance as the CACNA1D founder variant is only observed in families from the Khyber Pakhtunkhwa (KPK) province, in Pakistan. Therefore, screening patients with congenital deafness for SAN dysfunction in this province could ensure adequate follow-up and prevent cardiac failure associated with SAN.

Novel digenic inheritance of PCDH15 and USH1G underlies profound non-syndromic hearing impairment.

BACKGROUND: Digenic inheritance is the simplest model of oligenic disease. It can be observed when there is a strong epistatic interaction between two loci. For both syndromic and non-syndromic hearing impairment, several forms of digenic inheritance have been reported. METHODS: We performed exome sequencing in a Pakistani family with profound non-syndromic hereditary hearing impairment to identify the genetic cause of disease. RESULTS: We found that this family displays digenic inheritance for two trans heterozygous missense mutations, one in PCDH15 [p.(Arg1034His)] and another in USH1G [p.(Asp365Asn)]. Both of these genes are known to cause autosomal recessive non-syndromic hearing impairment and Usher syndrome. The protein products of PCDH15 and USH1G function together at the stereocilia tips in the hair cells and are necessary for proper mechanotransduction. Epistasis between Pcdh15 and Ush1G has been previously reported in digenic heterozygous mice. The digenic mice displayed a significant decrease in hearing compared to age-matched heterozygous animals. Until now no human examples have been reported. CONCLUSIONS: The discovery of novel digenic inheritance mechanisms in hereditary hearing impairment will aid in understanding the interaction between defective proteins and further define inner ear function and its interactome.

Recessive progressive symmetric erythrokeratoderma results from a homozygous loss-of-function mutation of KRT83 and is allelic with dominant monilethrix.

BACKGROUND: Progressive symmetric erythrokeratoderma (PSEK) is a rare skin disorder characterised by symmetrically distributed demarcated hyperkeratotic plaques, often with associated palmoplantar hyperkeratosis, with new plaques appearing over time. Most cases are inherited in an autosomal dominant manner, although a few cases exhibit apparent autosomal recessive inheritance. OBJECTIVE: To identify the gene underlying autosomal recessive PSEK in a large Pakistani kindred. METHODS: We first carried out autozygosity mapping using microsatellite markers in candidate regions of the genome. We then carried out exome sequencing of five family members, autozygosity mapping and mutation analysis using the exome data and verification by Sanger sequencing. RESULTS: Autozygosity mapping and exome sequencing identified a homozygous frameshift deletion (c.811delA; p.Ser271fs) in KRT83, which co-segregated with the PSEK phenotype in the family and which is expected to abolish keratin 83, a type II keratin of hair and skin. CONCLUSIONS: At least some cases of PSEK result from loss-of-function mutations in KRT83. Heterozygous missense substitutions in KRT83 have been implicated in autosomal dominant monilethrix, a rare hair disorder. Our findings indicate that at least some cases of autosomal recessive PSEK and autosomal dominant monilethrix are allelic, respectively resulting from loss-of-function and missense mutations in the KRT83 gene. Together, these findings indicate that different types of mutations in KRT83 can result in quite different skin and hair phenotypes.

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.

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

PURPOSE: To investigate the molecular basis of Bardet-Biedl syndrome (BBS) in five consanguineous families of Pakistani origin. METHODS: Linkage in two families (A and B) was established to BBS7 on chromosome 4q27, in family C to BBS8 on chromosome 14q32.1, and in family D to BBS10 on chromosome 12q21.2. Family E was investigated directly with exome sequence analysis. RESULTS: Sanger sequencing revealed two novel mutations and three previously reported mutations in the BBS genes. These mutations include two deletions (c.580_582delGCA, c.1592_1597delTTCCAG) in the BBS7 gene, a missense mutation (p.Gln449His) in the BBS8 gene, a frameshift mutation (c.271_272insT) in the BBS10 gene, and a nonsense mutation (p.Ser40*) in the MKKS (BBS6) gene. CONCLUSIONS: Two novel mutations and three previously reported variants, identified in the present study, further extend the body of evidence implicating BBS6, BBS7, BBS8, and BBS10 in causing BBS.

Mitral regurgitation as a phenotypic manifestation of nonphotosensitive trichothiodystrophy due to a splice variant in MPLKIP.

BACKGROUND: Nonphotosensitive trichothiodystrophy (TTDN) is a rare autosomal recessive disorder of neuroectodermal origin. The condition is marked by hair abnormalities, intellectual impairment, nail dystrophies and susceptibility to infections but with no UV sensitivity. METHODS: We identified three consanguineous Pakistani families with varied TTDN features and used homozygosity mapping, linkage analysis, and Sanger and exome sequencing in order to identify pathogenic variants. Haplotype analysis was performed and haplotype age estimated. A splicing assay was used to validate the effect of the MPLKIP splice variant on expression. RESULTS: Affected individuals from all families exhibit several TTDN features along with a heart-specific feature, i.e. mitral regurgitation. Exome sequencing in the probands from families ED168 and ED241 identified a homozygous splice mutation c.339 + 1G > A within MPLKIP. The same splice variant co-segregates with TTDN in a third family ED210. The MPLKIP splice variant was not found in public databases, e.g. the Exome Aggregation Consortium, and in unrelated Pakistani controls. Functional analysis of the splice variant confirmed intron retention, which leads to protein truncation and loss of a phosphorylation site. Haplotype analysis identified a 585.1-kb haplotype which includes the MPLKIP variant, supporting the existence of a founder haplotype that is estimated to be 25,900 years old. CONCLUSION: This study extends the allelic and phenotypic spectra of MPLKIP-related TTDN, to include a splice variant that causes cardiomyopathy as part of the TTDN phenotype.

The potential toxicity of chemically fabricated silver nanomaterials based on accumulation and histological changes in fish (Cyprinus carpio).

The bio-reductive fabrication of nanomaterials is a developing arena of study that seeks to fabricate nanoparticles (NPs) using microorganisms, plants, and animal blood. However, the chemical approach of AgNPs fulfills the need of abundant need of NPs. In contrast, chemically fabricated AgNPs are more toxic than biological AgNPs. Therefore, the current study aimed to assess and evaluate the chemically fabricated silver nanoparticles (AgNPs) for their possible toxicity in Common carp fish (Cyprinus carpio). The chemically synthesized silver nanoparticles were purchased from the market and applied for their possible toxicity. The chemically fabricated AgNPs were used against the Cyprinus carpio for bioaccumulation in different organs and histological alterations in the intestine and muscles. The results revealed that the AgNPs were mostly accumulated in the intestines followed by the gills, liver, and muscles (p < .05). The accumulated AgNPs caused histological alterations in gills and intestines at the highest concentration (0.08 mg/L). However, no alterations were observed by the middle and lowest concentration of AgNPs, particularly, in the intestine. In conclusion, more extensive research is required to establish the hazards related to the use of nanoparticles to disclose their negative effects on fish and the aquatic environment. REASEARCH HIGHLIGHTS: The chemical method fabricates a large amount of AgNPs Additionally, considered more toxic than the bio-reductive method AgNPs have excellent and diverse applications AgNPs deposited in various organs and cause histological changes.

Homozygous Missense Variant in the Solute Carrier Organic Anion Transporter 2A1 (SLCO2A1) Gene Underlies Isolated Nail Clubbing.

BACKGROUND: Inherited isolated nail clubbing is a very rare Mendelian condition in humans, characterized by enlargement of the terminal segments of fingers and toes with thickened nails. Mutations in two genes have been reported to cause isolated nail clubbing in humans, which are the SLCO2A1 gene and the HPGD gene. OBJECTIVES: An extended Pakistani family having two affected siblings born of unaffected consanguineous union was included in the study. Predominant isolated congenital nail clubbing (ICNC) without any other systemic abnormalities was observed, which we aimed to characterize at clinico-genetic level. METHODS: Whole exome coupled with Sanger sequencing were employed to uncover the sequence variant as a cause of the disease. Furthermore, protein modeling was carried out to reveal the predicted possible effect of the mutation at the protein level. RESULTS: Whole exome sequencing data analysis revealed a novel biallelic sequence variant (c.155T>A; p.Phe52Tyr) in the SLCO2A1 gene. Further, Sanger sequencing analysis validated and confirmed the segregation of the novel variant in the entire family. Subsequently, protein modeling of the wild-type and mutated SLCO2A1 revealed broad-scale change, which might compromise the proteins' secondary structure and function. CONCLUSION: The present study adds another mutation to the SLCO2A1-related pathophysiology. The involvement of SLCO2A1 in the pathogenesis of ICNC may open exciting perceptions of this gene in nail development/morphogenesis.

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.

Association of Somatic ATP2A2 Damaging Variants With Grover Disease.

Importance: Grover disease (GD), a truncal eruption that typically occurs in older individuals, is exacerbated by sweating, irradiation, cancers, medications, kidney failure, and organ transplantation. The pathobiology of GD remains unknown. Objective: To determine if damaging somatic single-nucleotide variants (SNVs) are associated with GD. Design, Setting, and Participants: In this retrospective case series, we identified consecutive patients from a dermatopathology archive over a 4-year period (January 2007 to December 2011) who had 1 biopsy with a clinical diagnosis of GD confirmed via histopathologic findings and another non-GD biopsy. Participant DNA was extracted from both biopsy tissues and sequenced to high depth with a 51-gene panel to screen for SNVs in genes previously associated with acantholysis and Mendelian disorders of cornification. Analysis took place between 2021 and 2023. Main Outcomes and Measures: Comparative analysis of sequencing data from paired GD and control tissue was employed to identify SNVs predicted to affect gene function, which were exclusive to, or highly enriched in, GD tissue. Results: Overall, 12 of 15 cases of GD (12 men and 3 women; mean [SD] age, 68.3 [10.0] years) were associated with C>T or G>A ATP2A2 SNVs in GD tissue; all were predicted to be highly damaging via combined annotation dependent depletion (CADD) scores, and 4 were previously associated with Darier disease. In 9 cases (75%), the GD-associated ATP2A2 SNV was absent from control tissue DNA, and in 3 cases (25%), ATP2A2 SNVs were enriched 4- to 22-fold in GD vs control tissue. Conclusions and Relevance: In this case series study of 15 patients, damaging somatic ATP2A2 SNVs were associated with GD. This discovery expands the spectrum of acantholytic disorders associated with ATP2A2 SNVs and highlights the role of somatic variation in acquired disorders.

Exploring the natural products chemical space to abrogate the F3L-dsRNA interface of monkeypox virus to enhance the immune responses using molecular screening and free energy calculations.

Amid the ongoing monkeypox outbreak, there is an urgent need for the rapid development of effective therapeutic interventions capable of countering the immune evasion mechanisms employed by the monkeypox virus (MPXV). The evasion strategy involves the binding of the F3L protein to dsRNA, resulting in diminished interferon (IFN) production. Consequently, our current research focuses on utilizing virtual drug screening techniques to target the RNA binding domain of the F3L protein. Out of the 954 compounds within the South African natural compound database, only four demonstrated notable docking scores: -6.55, -6.47, -6.37, and -6.35 kcal/mol. The dissociation constant (KD) analysis revealed a stronger binding affinity of the top hits 1-4 (-5.34, -5.32, -5.29, and -5.36 kcal/mol) with the F3L in the MPXV. All-atom simulations of the top-ranked hits 1 to 4 consistently exhibited stable dynamics, suggesting their potential to interact effectively with interface residues. This was further substantiated through analyses of parameters such as radius of gyration (Rg), Root Mean Square Fluctuation, and hydrogen bonding. Cumulative assessments of binding free energy confirmed the top-performing candidates among all the compounds, with values of -35.90, -52.74, -28.17, and -32.11 kcal/mol for top hits 1-4, respectively. These results indicate that compounds top hit 1-4 could hold significant promise for advancing innovative drug therapies, suggesting their suitability for both in vivo and in vitro experiments.

A novel homozygous missense variant p.D339N in the PKLR gene correlates with pyruvate kinase deficiency in a Pakistani family: a case report.

BACKGROUND: Pyruvate kinase deficiency is an exceptionally rare autosomal recessive Mendelian disorder caused by bi-allelic pathogenic variants in the PKLR gene. It is mainly characterized by chronic nonspherocytic hemolytic anemia though other symptoms such as splenomegaly, hepatomegaly, pallor, fatigue, iron overload, shortness of breath, hyperbilirubinemia, and gallstones might also prevail. CASE PRESENTATION: We present here a novel genetic defect in the PKLR gene that correlates with pyruvate kinase deficiency phenotype in a consanguineous family from North-Western Pakistan. The family included three affected individuals who were all born to consanguineous parents. The proband, a 13-year-old female of Pashtun ethnicity, showed chronic nonautoimmune hemolytic anemia since birth, extremely low hemoglobin (7.6 g/dL) and pyruvate kinase (12.4 U/g Hb) levels, splenomegaly, and hepatomegaly. Bone marrow aspirate showed a markedly decreased myeloid to erythroid ratio and hypercellular marrow particles due to hyperplasia of the erythroid elements. Molecular characterization of the proband's genomic DNA uncovered a likely pathogenic homozygous missense variant p.[D339N] in exon 7 of the PKLR gene. In-depth in silico analysis and familial cosegregation implies p.[D339N] as the likely cause of pyruvate kinase deficiency in this family. Further in vitro or in vivo studies are required to validate the impact of p.[D339N] on protein structure and/or stability, and to determine its role in the disease pathophysiology. CONCLUSIONS: In summary, these findings suggest a novel genetic defect in the PKLR gene as a likely cause of pyruvate kinase deficiency, thus further expanding the mutational landscape of this rare Mendelian disorder.