A novel homozygous missense TTC12 variant identified in an infertile Pakistani man with severe oligoasthenoteratozoospermia and primary ciliary dyskinesia.

TTC12 is a cytoplasmic and centromere-localized protein that plays a role in the proper assembly of dynein arm complexes in motile cilia in both respiratory cells and sperm flagella. This finding underscores its significance in cellular motility and function. However, the wide role of TTC12 in human spermatogenesis-associated primary ciliary dyskinesia (PCD) still needs to be elucidated. Whole-exome sequencing (WES) and Sanger sequencing were performed to identify potentially pathogenic variants causing PCD and multiple morphological abnormalities of sperm flagella (MMAF) in an infertile Pakistani man. Diagnostic imaging techniques were used for PCD screening in the patient. Real-time polymerase chain reaction (RT‒PCR) was performed to detect the effect of mutations on the mRNA abundance of the affected genes. Papanicolaou staining and scanning electron microscopy (SEM) were carried out to examine sperm morphology. Transmission electron microscopy (TEM) was performed to examine the ultrastructure of the sperm flagella, and the results were confirmed by immunofluorescence staining. Using WES and Sanger sequencing, a novel homozygous missense variant (c.C1069T; p.Arg357Trp) in TTC12 was identified in a patient from a consanguineous family. A computed tomography scan of the paranasal sinuses confirmed the symptoms of the PCD. RT-PCR showed a decrease in TTC12 mRNA in the patient's sperm sample. Papanicolaou staining, SEM, and TEM analysis revealed a significant change in shape and a disorganized axonemal structure in the sperm flagella of the patient. Immunostaining assays revealed that TTC12 is distributed throughout the flagella and is predominantly concentrated in the midpiece in normal spermatozoa. In contrast, spermatozoa from patient deficient in TTC12 showed minimal staining intensity for TTC12 or DNAH17 (outer dynein arms components). This could lead to MMAF and result in male infertility. This novel TTC12 variant not only illuminates the underlying genetic causes of male infertility but also paves the way for potential treatments targeting these genetic factors. This study represents a significant advancement in understanding the genetic basis of PCD-related infertility.

Analysis of a non-lethal biallelic frameshift mutation in ZMPSTE24 reveals utilization of alternative translation initiation codons.

Restrictive dermopathy (RD) is a lethal condition caused by biallelic loss-of-function mutations in ZMPSTE24, whereas mutations preserving residual enzymatic activity of the ZMPSTE24 protein lead to the milder mandibuloacral dysplasia with type B lipodystrophy (MADB) phenotype. Remarkably, we identified a homozygous, presumably loss-of-function mutation in ZMPSTE24 [c.28_29insA, p.(Leu10Tyrfs*37)] in two consanguineous Pakistani families segregating MADB. To clarify how lethal consequences are prevented in affected individuals, functional analysis was performed. Expression experiments supported utilization of two alternative translation initiation sites, preventing complete loss of protein function consistent with the relatively mild phenotypic outcome in affected patients. One of these alternative start codons is newly formed at the insertion site. Our findings indicate that the creation of new potential start codons through N-terminal mutations in other disease-associated genes should generally be taken into consideration in the variant interpretation process.

Identification of Four Novel Candidate Genes for Non-syndromic Intellectual Disability in Pakistani Families.

Intellectual disability, a genetically and clinically varied disorder and is a significant health problem, particularly in less developed countries due to larger family size and high ratio of consanguineous marriages. In the current genetic study, we investigate and find the novel disease causative factors in the four Pakistani families with severe type of non-syndromic intellectual disability. For genetic analysis whole-exome sequencing (WES) and Sanger sequencing was performed. I-TASSER and Cluspro tools were used for Protein modeling and Protein-protein docking. Sanger sequencing confirms the segregation of novel homozygous variants in all the families i.e., c.245 T > C; p.Leu82Pro in SLC50A1 gene in family 1, missense variant c.1037G > A; p.Arg346His in TARS2 gene in family 2, in family 3 and 4, nonsense mutation c.234G > A; p.Trp78Term and missense mutation c.2200G > A; p.Asp734Asn in TBC1D3 and ANAPC2 gene, respectively. In silico functional studies have found the drastic effect of these mutations on protein structure and its interaction properties. Substituted amino acids were highly conserved and present on highly conserved region throughout the species. The discovery of pathogenic variants in SLC50A1, TARS2, TBC1D1 and ANAPC2 shows that the specific pathways connected with these genes may be important in cognitive impairment. The decisive role of pathogenic variants in these genes cannot be determined with certainty due to lack of functional data. However, exome sequencing and segregation analysis of all filtered variants revealed that the currently reported variants were the only variations from the respective families that segregated with the phenotype in the family.

The molecular genetics of human appendicular skeleton.

Disorders that result from de-arrangement of growth, development and/or differentiation of the appendages (limbs and digit) are collectively called as inherited abnormalities of human appendicular skeleton. The bones of appendicular skeleton have central role in locomotion and movement. The different types of appendicular skeletal abnormalities are well described in the report of "Nosology and Classification of Genetic skeletal disorders: 2019 Revision". In the current article, we intend to present the embryology, developmental pathways, disorders and the molecular genetics of the appendicular skeletal malformations. We mainly focused on the polydactyly, syndactyly, brachydactyly, split-hand-foot malformation and clubfoot disorders. To our knowledge, only nine genes of polydactyly, five genes of split-hand-foot malformation, nine genes for syndactyly, eight genes for brachydactyly and only single gene for clubfoot have been identified to be involved in disease pathophysiology. The current molecular genetic data will help life sciences researchers working on the rare skeletal disorders. Moreover, the aim of present systematic review is to gather the published knowledge on molecular genetics of appendicular skeleton, which would help in genetic counseling and molecular diagnosis.

Mutation screening of multiple Pakistani MCPH families revealed novel and recurrent protein-truncating mutations of ASPM.

Autosomal primary microcephaly (MCPH) is a heterogenetic disorder that affects brain's cerebral cortex size and leads to a reduction in the cranial vault. Along with the hallmark feature of reduced head circumference, microcephalic patients also exhibit a variable degree of intellectual disability as well. Genetic studies have reported 28 MCPH genes, most of which produce microtubule-associated proteins and are involved in cell division. Herein this study, 14 patients from seven Pashtun origin Pakistani families of primary microcephaly were analyzed. Mutation analysis was performed through targeted Sanger DNA sequencing on the basis of phenotype-linked genetic makeup. Genetic analysis in one family found a novel pathogenic DNA change in the abnormal spindle microtubule assembly (ASPM) gene (NM_018136.4:c.3871dupGA), while the rest of the families revealed recurrent nonsense mutation c.3978G>A (p.Trp1326*) in the same gene. The novel reported frameshift insertion presumably truncates the protein p.(Lys1291Glyfs*14) and deletes the N-terminus domains. Identification of novel ASPM-truncating mutation expands the mutational spectrum of the ASPM gene, while mapping of recurrent mutation c.3978G>A (p.Trp1326*) will aid in establishing its founder effect in the Khyber Pakhtunkhwa (KPK) inhabitant population of Pakistan and should be suggestively screened for premarital counseling of MCPH susceptible families. Most of the recruited families are related to first-degree consanguinity. Hence, all the family elders were counseled to avoid intrafamilial marriages.

A novel protein truncating mutation in L2HGDH causes L-2-hydroxyglutaric aciduria in a consanguineous Pakistani family.

BACKGROUND: L-2-hydroxyglutaric aciduria (L2HGA) is a rare neurometabolic disorder that occurs due to accumulation of L-2-hydroxyglutaric acid in the cerebrospinal fluid (CSF), plasma and urine. The clinical manifestation of L2HGA includes intellectual disability, cerebellar ataxia, epilepsy, speech problems and macrocephaly. METHODS: In the present study, we ascertained a multigenerational consanguineous Pakistani family with 5 affected individuals. Clinical studies were performed through biochemical tests and brain CT scan. Locus mapping was carried out through genome-wide SNP genotyping, whole exome sequencing and Sanger sequencing. For in silico studies protein structural modeling and docking was done using I-TASSER, Cluspro and AutoDock VINA tools. RESULTS: Affected individuals presented with cognitive impairment, gait disturbance, speech difficulties and psychomotor delay. Radiologic analysis of a male patient revealed leukoaraiosis with hypoattenuation of cerebral white matter, suggestive of hypomyelination. Homozygosity mapping in this family revealed a linkage region on chromosome 14 between markers rs2039791 and rs781354. Subsequent whole exome analysis identified a novel frameshift mutation NM_024884.3:c.180delG, p.(Ala62Profs*24) in the second exon of L2HGDH. Sanger sequencing confirmed segregation of this mutation with the disease phenotype. The identification of the most N-terminal loss of function mutation published thus far further expands the mutational spectrum of L2HGDH.

Alopecia-mental retardation syndrome: Molecular genetics of a rare neuro-dermal disorder.

Alopecia-mental retardation syndrome (APMR) is a rare autosomal recessive neuro-dermal disorder. It is characterized by heterogeneous phenotypic features, that is, absence of hair on the scalp, eyelashes, and eyebrows and mild to severe intellectual disability. So far, approximately 14 families (i.e., Iranian, Pakistani, and Swiss) with APMR have been reported in the scientific literature. Its precise prevalence is still unknown, but according to a predictive estimate, it prevails with the ratio of 1 in 1,000,000 persons worldwide. Until now, only four loci (two characterized and two uncharacterized) have been reported to be involved in APMR. The pathogenic variants in alpha-2-HS-glycoprotein [AHSG; APMR1 (MIM#203650)] and lanosterol synthase [LSS; APMR4 (MIM#618840)] are the characterized genetic factors associated with APMR. Among them, AHSG was reported in a consanguineous Iranian family and LSS gene in a Swiss origin family, while the remaining two uncharacterized loci, that is, APMR2 and APMR3, are reported in the Pakistani population. The current mini-report discusses the molecular genetics and mutational spectrum of APMR syndrome, its differential diagnosis from related disorders, and prediction of plausible candidate genes in two uncharacterized loci.

The molecular genetics of UV-Sensitive syndrome: A rare dermal anomaly.

Ultraviolet-sensitive syndrome is a rare skin disorder characterised by heterogeneous phenotypic spectrum of skin freckling, telangiectasia and acute sunburn. It usually has an autosomal recessive pattern. So far, only 18 patients from nine different families (Japanese, French, Israeli, Iranian and Pakistani) have been reported in scientific literature. Its precise prevalence is still unknown, but, according to an estimate, its prevalence ratio is 1:100,000 worldwide. Until now, only three genes have been reported to be involved in the syndrome; the Excision Repair Cross-Complementing, Group 6, the Excision Repair Cross-Complementing, Group 8 and the UV-Stimulated Scaffold Protein A (UVSSA). Among these genes, the last one is reported to be more prevalent among different ethnicities, including Pakistani. Physiologically, most of the syndrome genes are involved in the transcription-coupled nucleotide excision pathway. In order to reduce the disease severity, the patients are advised to use medicated skin moisturisers or sun-blocks, sunglasses and gloves, while going out in the sun to avoid sun exposure. The current narrative review was planned to discuss the molecular genetics and the mutational spectrum of the syndrome, and to describe the differential diagnosis of various related disorders in order to facilitate clinical researchers.

Expression analysis of tumour necrosis factor alpha (TNF-α) and alkaline phosphatase in occupational workers exposed to low dose of X-radiation: A case-control study.

OBJECTIVES: To evaluate liver and inflammatory biomarkers in occupationally exposed radiology workers. METHODS: The descriptive study was conducted at Mufti Mehmood Memorial Teaching Hospital and Gomal Centre of Biochemistry and Biotechnology, Gomal University, Dera Ismail Khan, Pakistan, from September 2017 to May 2018, and comprised X-ray technicians working 48-72 hours per week, and a group of age- and gender-matched unexposed healthy controls. The exposed group was divided into three sub-groups based on their radiation work duration. Liver health status involved estimation of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma-glutamyl transferase GGT and bilirubin through automated chemistry analyser, while serum tumour necrosis factor-alpha and interleukin- 6 levels through enzyme-linked immunosorbent assay technique. Relative gene expression analysis of tumour necrosis factor-alpha and alkaline phosphatase was performed through reverse transcription-polymerase chain reaction. Data was analysed using SPSS 20. RESULTS: Of the 70 subjects, 50(71.4%) were cases with a mean age of 36.98±8.07 years and 20(28.6%) were controls with a mean age of 36.80±7.78 years. Serum alanine aminotransferase and alkaline phosphatase levels showed significant elevation in the cases compared to the controls (p<0.0001), although alanine aminotransferase levels were within the normal range. The difference in aspartate aminotransferase, gamma-glutamyl transferase and bilirubin levels was not significant (p>0.05). Tumour necrosis factor-alpha concentration was significantly high in the cases compared to the controls (p<0.0001). In contrast with proteomic analysis, relative gene expression analysis revealed reduced level of alkaline phosphatase and tumour necrosis factor-alpha in the cases compared to the controls (p<0.05). CONCLUSIONS: Serum proteomic analysis of X-ray technicians indicated acute inflammatory conditions, while genomic analysis exhibited down-regulation of alkaline phosphatase and tumour necrosis factor-alpha genes.

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.

Genetic studies of multiple consanguineous Pakistani families segregating oculocutaneous albinism identified novel and reported mutations.

Oculocutaneous albinism (OCA) is an autosomal-recessive disorder of a defective melanin pathway. The condition is characterized by hypopigmentation of hair, dermis, and ocular tissue. Genetic studies have reported seven nonsyndromic OCA genes, among which Pakistani OCA families mostly segregate TYR and OCA2 gene mutations. Here in the present study, we investigate the genetic factors of eight consanguineous OCA families from Pakistan. Genetic analysis was performed through single-nucleotide polymorphism (SNP) genotyping (for homozygosity mapping), whole exome sequencing (for mutation identification), Sanger sequencing (for validation and segregation analysis), and quantitative PCR (qPCR) (for copy number variant [CNV] validation). Genetic mapping in one family identified a novel homozygous deletion mutation of the entire TYRP1 gene, and a novel deletion of exon 19 in the OCA2 gene in two apparently unrelated families. In three further families, we identified homozygous mutations in TYR (NM_000372.4:c.1424G > A; p.Trp475*), NM_000372.4:c.895C > T; p.Arg299Cys), and SLC45A2 (NM_016180:c.1532C > T; p.Ala511Val). For the remaining two families, G and H, compound heterozygous TYR variants NM_000372.4:c.1037-7T > A, NM_000372.4:c.1255G > A (p.Gly419Arg), and NM_000372.4:c.1255G > A (p.Gly419Arg) and novel variant NM_000372.4:c.248T > G; (p.Val83Gly), respectively, were found. Our study further extends the evidence of TYR and OCA2 as genetic mutation hot spots in Pakistani families. Genetic screening of additional OCA cases may also contribute toward the development of Pakistani specific molecular diagnostic tests, genetic counseling, and personalized healthcare.

Genetic study of Khyber-Pukhtunkhwa resident Pakistani families presenting primary microcephaly with intellectual disability.

OBJECTIVE: To investigate the genetic factor responsible for causing microcephaly and determine allelic heterogeneity of Abnormal spindle microtubule gene. METHODS: The genetic study was conducted at the Kohat University of Science and Technology, Kohat, and Gomal University, D.I.Khan, Pakistan, during 2017-18, and comprised 5 consanguineous families from South Waziristan, Kurram Agency, Karak, Bannu and Dera Ismail Khan regions of the country's Khyber Pakhtukhwa province. Blood samples from all available and cooperative family members (including normal and affected) were obtained, and molecular analysis was carried out through whole genome single nucleotide polymorphisms genotyping, exome sequencing and Sanger sequencing. RESULTS: Of the 15 patients, 9(60%) were males and 6(40%) were females. Genetic mapping revealed linkage to the MCPH5 locus which harbours the microcephaly-associated abnormal spindle-like microcephaly gene. Mutation analysis of the gene identified missense mutation c.3978G>A (p.Trp1326*) in families A, B and C, a deletion mutation c.7782_7783delGA (p.(Lys2595Serfs*6)) in family D, and a splice site defect c.2936+5G>A in family E. CONCLUSIONS: There was suggestion of strong founder effect of mutation c.3978G>A (p.Trp1326*).

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.

Ophthalmo-genetic analysis of Pakistani patients with nonsyndromic oculocutaneous albinism through whole exome sequencing.

Oculocutaneous albinism (OCA) is a disorder of defective melanin biosynthesis that is characterized by hypo-pigmentation of skin, hair and retinal pigment epithelium. Phenotypically, OCA patients exhibit white milky skin, whitish to golden hair and deterioration of retinal cells. Until recently, genetic studies have reported seven causative genes (TYR, TYRP1, OCA2, SLC45A2, SLC24A2, C10ORF11 and MCIR) and an uncharacterized OCA5 locus. Herein we present the medico-genetic study of three Pakistani patients inheriting autosomal recessive OCA. Whole exome sequencing, followed by Sanger DNA sequencing for segregation analysis, revealed recurrent mutations c.346C>T (p.Arg116*) and c.1255G>A (p.Gly419Arg) (family A and B respectively) in TYR gene, while the patient from family C did not reveal any known gene mutation, which suggests the involvement of some novel genetic factor. It is the first report of mapping c.346C>T mutation in a Pakistani patient. Our study further extends the evidence of genetic hotspots regions in TYR gene causing OCA in Pakistani population.

The Molecular Genetics of Autosomal Recessive Nonsyndromic Intellectual Disability: a Mutational Continuum and Future Recommendations.

Intellectual disability (ID) is a clinical manifestation of the central nervous system without any major dysmorphologies of the brain. Biologically it affects learning capabilities, memory, and cognitive functioning. The basic defining features of ID are characterized by IQ<70, age of onset before 18 years, and impairment of at least two of the adaptive skills. Clinically it is classified in a syndromic (with additional abnormalities) and a nonsyndromic form (with only cognitive impairment). The study of nonsyndromic intellectual disability (NSID) can best explain the pathophysiology of cognition, intelligence and memory. Genetic analysis in autosomal recessive nonsyndrmic ID (ARNSID) has mapped 51 disease loci, 34 of which have revealed their defective genes. These genes play diverse physiological roles in various molecular processes, including methylation, proteolysis, glycosylation, signal transduction, transcription regulation, lipid metabolism, ion homeostasis, tRNA modification, ubiquitination and neuromorphogenesis. High-density SNP array and whole exome sequencing has increased the pace of gene discoveries and many new mutations are being published every month. The lack of uniform criteria has assigned multiple identifiers (or accession numbers) to the same MRT locus (e.g. MRT7 and MRT22). Here in this review we describe the molecular genetics of ARNSID, prioritize the candidate genes in uncharacterized loci, and propose a new nomenclature to reorganize the mutation data that will avoid the confusion of assigning duplicate accession numbers to the same ID locus and to make the data manageable in the future as well.

Homozygosity mapping identified a novel protein truncating mutation (p.Ser100Leufs*24) of the BBS9 gene in a consanguineous Pakistani family with Bardet Biedl syndrome.

BACKGROUND: Bardet Biedl Syndrome (BBS) is a rare condition of multi-organ dysfunction with characteristic clinical features of retinal degeneration, truncal obesity, postaxial polydactyly, genital anomaly, intellectual disability and renal dysfunction. It is a hetero-genetic disorder and nineteen BBS genes have been discovered so far. METHODS: Whole genome SNP genotyping was performed by using CytoScan® 750 K array (Affymetrix). Subsequently, the segregation of the disease locus in the whole family was carried out by genotyping STS markers within the homozygous interval. Finally, the mutation analysis was performed by Sanger DNA sequencing. RESULTS: In the present molecular study a consanguineous Pakistani family, with autosomal recessive BBS, was analyzed. The clinical analysis of affected individuals presented with synpolydactyly, obesity, intellectual disability, renal abnormality and retinitis pigmentosa. The presented phenotype was consistent with the major features of BBS syndrome. Homozygosity mapping identified a common homozygous interval within the known BBS9 locus. Sequence analysis of BBS9/PTHB1 gene revealed a single base deletion of c.299delC (p.Ser100Leufs*24) in exon 4. This frame-shift mutation presumably leads to a 122 amino acid truncated protein with complete loss of its C-terminal PTHB1 domain in combination with a partial loss of the N-terminal PTHB1 domain as well. BBS9/PTHB1 gene mutations have been shown to be associated with BBS syndrome and to the best of our knowledge this study reports the first Pakistani family linked to the BBS9 gene. CONCLUSION: Our molecular findings expand the mutational spectrum of BBS9 gene and also explain the genetic heterogeneity of Pakistan families with BBS syndrome. The growing number of mutations in BBS genes in combination with a detailed phenotypical description of patients will be helpful for genotype-phenotype correlation, targeted genetic diagnosis, prenatal screening and carrier testing of familial and non-familial BBS patients.

Genetic analysis of consanguineous families presenting with congenital ocular defects.

Anophthalmia and microphthalmia (A/M) are a group of rare developmental disorders that affect the size of the ocular globe. A/M may present as the sole clinical feature, but are also frequently found in a variety of syndromes. A/M is genetically heterogeneous and can be caused by chromosomal aberrations, copy number variations and single gene mutations. To date, A/M has been caused by mutations in at least 20 genes that show different modes of inheritance. In this study, we enrolled eight consanguineous families with A/M, including seven from Pakistan and one from India. Sanger and exome sequencing of DNA samples from these families identified three novel mutations including two mutations in the Aldehyde Dehydrogenase 1 Family Member A3 (ALDH1A3) gene, [c.1310_1311delAT; p.(Tyr437Trpfs*44) and c.964G > A; p.(Val322Met)] and a single missense mutation in Forkhead Box E3 (FOXE3) gene, [c.289A > G p.(Ile97Val)]. Additionally two previously reported mutations were identified in FOXE3 and in Visual System Homeobox 2 (VSX2). This is the first comprehensive study on families with A/M from the Indian subcontinent which provides further evidence for the involvement of known genes with novel and recurrent mutations.

Mutation in NSUN2, which encodes an RNA methyltransferase, causes autosomal-recessive intellectual disability.

Causes of autosomal-recessive intellectual disability (ID) have, until very recently, been under researched because of the high degree of genetic heterogeneity. However, now that genome-wide approaches can be applied to single multiplex consanguineous families, the identification of genes harboring disease-causing mutations by autozygosity mapping is expanding rapidly. Here, we have mapped a disease locus in a consanguineous Pakistani family affected by ID and distal myopathy. We genotyped family members on genome-wide SNP microarrays and used the data to determine a single 2.5 Mb homozygosity-by-descent (HBD) locus in region 5p15.32-p15.31; we identified the missense change c.2035G>A (p.Gly679Arg) at a conserved residue within NSUN2. This gene encodes a methyltransferase that catalyzes formation of 5-methylcytosine at C34 of tRNA-leu(CAA) and plays a role in spindle assembly during mitosis as well as chromosome segregation. In mouse brains, we show that NSUN2 localizes to the nucleolus of Purkinje cells in the cerebellum. The effects of the mutation were confirmed by the transfection of wild-type and mutant constructs into cells and subsequent immunohistochemistry. We show that mutation to arginine at this residue causes NSUN2 to fail to localize within the nucleolus. The ID combined with a unique profile of comorbid features presented here makes this an important genetic discovery, and the involvement of NSUN2 highlights the role of RNA methyltransferase in human neurocognitive development.

Mutations in the alpha 1,2-mannosidase gene, MAN1B1, cause autosomal-recessive intellectual disability.

We have used genome-wide genotyping to identify an overlapping homozygosity-by-descent locus on chromosome 9q34.3 (MRT15) in four consanguineous families affected by nonsyndromic autosomal-recessive intellectual disability (NS-ARID) and one in which the patients show additional clinical features. Four of the families are from Pakistan, and one is from Iran. Using a combination of next-generation sequencing and Sanger sequencing, we have identified mutations in the gene MAN1B1, encoding a mannosyl oligosaccharide, alpha 1,2-mannosidase. In one Pakistani family, MR43, a homozygous nonsense mutation (RefSeq number NM_016219.3: c.1418G>A [p.Trp473*]), segregated with intellectual disability and additional dysmorphic features. We also identified the missense mutation c. 1189G>A (p.Glu397Lys; RefSeq number NM_016219.3), which segregates with NS-ARID in three families who come from the same village and probably have shared inheritance. In the Iranian family, the missense mutation c.1000C>T (p.Arg334Cys; RefSeq number NM_016219.3) also segregates with NS-ARID. Both missense mutations are at amino acid residues that are conserved across the animal kingdom, and they either reduce k(cat) by ∼1300-fold or disrupt stable protein expression in mammalian cells. MAN1B1 is one of the few NS-ARID genes with an elevated mutation frequency in patients with NS-ARID from different populations.

A novel homozygous TSGA10 missense variant causes acephalic spermatozoa syndrome in a Pakistani family.

BACKGROUND: Acephalic spermatozoa syndrome is a rare type of teratozoospermia causing male infertility due to detachment of the sperm head and flagellum, which precludes fertilization potential. Although loss-of-function variations in several genes, including TSGA10, have been associated with acephalic spermatozoa syndrome, the genetic cause of many cases remains unclear. RESULTS: We recruited a Pakistani family with two infertile brothers who suffered from acephalic spermatozoa syndrome. Through whole-exome sequencing (WES) followed by Sanger sequencing, we identified a novel missense variant in TSGA10 (c.1112T > C, p. Leu371Pro), which recessively co-segregated with the acephalic spermatozoa syndrome within this family. Ultrastructural analyses of spermatozoa from the patient revealed that 98% of flagellar cross-sections displayed abnormal axonemal ultrastructure, in addition to the head-flagellum detachment. Real-time quantitative PCR analysis revealed almost no detectable TSAG10 mRNA and western blot analysis also failed to detect TSAG10 protein in patient's sperm samples while TSGA10 expression was clearly detected in control samples. Consistently, immunofluorescence analysis demonstrated the presence of TSGA10 signal in the midpiece of sperm from the control but a complete absence of TSGA10 signal in sperm from the patient. CONCLUSION: Altogether, our study identifies a novel TSGA10 pathogenic variant as a cause of acephalic spermatozoa syndrome in this family and provides information regarding the clinical manifestations associated with TSGA10 variants in human.

RéSUMé: CONTEXTE: Le syndrome des spermatozoïdes acéphaliques est un type rare de tératozoospermie provoquant une infertilité masculine en raison du détachement de la tête et du flagelle des spermatozoïdes, ce qui exclut une potentielle fécondation. Bien que des variations de perte de fonction dans plusieurs gènes, y compris TSGA10, aient été associées au syndrome des spermatozoïdes acéphaliques, la cause génétique de nombreux cas reste incertaine. RéSULTATS: Nous avons recruté une famille pakistanaise avec deux frères infertiles qui souffraient du syndrome des spermatozoïdes acéphaliques. Grâce au séquençage de l'exome entier (WES) suivi du séquençage Sanger, nous avons identifié un nouveau variant faux-sens dans TSGA10 (c.1112T > C, p. Leu371Pro), qui co-ségréguait de manière récessive avec le syndrome des spermatozoïdes acéphaliques au sein de cette famille. Les analyses ultrastructurales des spermatozoïdes des patients ont révélé que 98% des coupes transversales flagellaires présentaient une ultrastructure axonémiques anormales, en plus du décollement tête-flagelle. L'analyse quantitative par PCR en temps réel n'a révélé presque aucun ARNm TSAG10 détectable; l'analyse par transfert Western n'a pas non plus réussi à détecter la protéine TSAG10 dans les échantillons de sperme des patients, tandis que l'expression de TSGA10 a été clairement détectée dans les échantillons du témoin. De manière cohérente, l'analyse par immunofluorescence a démontré la présence du signal TSGA10 dans la partie médiane des spermatozoïdes du témoin, mais une absence totale de signal TSGA10 chez ceux des patients. CONCLUSION: Dans l'ensemble, notre étude identifie un nouveau variant pathogène de TSGA10 comme cause du syndrome des spermatozoïdes acéphaliques dans cette famille et fournit des informations concernant les manifestations cliniques associées aux variants de TSGA10 chez l'homme. MOTS-CLéS: Infertilité, TSGA10, Spermatozoïdes acéphaliques, Variations faux-sens.