Comparative analysis of in-silico tools in identifying pathogenic variants in dominant inherited retinal diseases.

Inherited retinal diseases (IRDs) are a group of rare genetic eye conditions that cause blindness. Despite progress in identifying genes associated with IRDs, improvements are necessary for classifying rare autosomal dominant (AD) disorders. AD diseases are highly heterogenous, with causal variants being restricted to specific amino acid changes within certain protein domains, making AD conditions difficult to classify. Here, we aim to determine the top-performing in-silico tools for predicting the pathogenicity of AD IRD variants. We annotated variants from ClinVar and benchmarked 39 variant classifier tools on IRD genes, split by inheritance pattern. Using area-under-the-curve (AUC) analysis, we determined the top-performing tools and defined thresholds for variant pathogenicity. Top-performing tools were assessed using genome sequencing on a cohort of participants with IRDs of unknown etiology. MutScore achieved the highest accuracy within AD genes, yielding an AUC of 0.969. When filtering for AD gain-of-function and dominant negative variants, BayesDel had the highest accuracy with an AUC of 0.997. Five participants with variants in NR2E3, RHO, GUCA1A, and GUCY2D were confirmed to have dominantly inherited disease based on pedigree, phenotype, and segregation analysis. We identified two uncharacterized variants in GUCA1A (c.428T>A, p.Ile143Thr) and RHO (c.631C>G, p.His211Asp) in three participants. Our findings support using a multi-classifier approach comprised of new missense classifier tools to identify pathogenic variants in participants with AD IRDs. Our results provide a foundation for improved genetic diagnosis for people with IRDs.

Novel Pathogenic Mutations Identified from Whole-Genome Sequencing in Unsolved Cases of Patients Affected with Inherited Retinal Diseases.

Inherited retinal diseases (IRDs) are a diverse set of visual disorders that collectively represent a major cause of early-onset blindness. With the reduction in sequencing costs in recent years, whole-genome sequencing (WGS) is being used more frequently, particularly when targeted gene panels and whole-exome sequencing (WES) fail to detect pathogenic mutations in patients. In this study, we performed mutation screens using WGS for a cohort of 311 IRD patients whose mutations were undetermined. A total of nine putative pathogenic mutations in six IRD patients were identified, including six novel mutations. Among them, four were deep intronic mutations that affected mRNA splicing, while the other five affected protein-coding sequences. Our results suggested that the rate of resolution of unsolved cases via targeted gene panels and WES can be further enhanced with WGS; however, the overall improvement may be limited.

A biallelic variant of DCAF13 implicated in a neuromuscular disorder in humans.

Neuromuscular disorders encompass a broad range of phenotypes and genetic causes. We investigated a consanguineous family in which multiple patients had a neuromuscular disorder characterized by a waddling gait, limb deformities, muscular weakness and facial palsy. Exome sequencing was completed on the DNA of three of the four patients. We identified a novel missense variant in DCAF13, ENST00000612750.5, NM_015420.7, c.907 G > A;p.(Asp303Asn), ENST00000616836.4, NM_015420.6, c.1363 G > A:p.(Asp455Asn) (rs1209794872) segregating with this phenotype; being homozygous in all four affected patients and heterozygous in the unaffected individuals. The variant was extremely rare in the public databases (gnomAD allele frequency 0.000007081); was absent from the DNA of 300 ethnically matched controls and affected an amino acid which has been conserved across 1-2 billion years of evolution in eukaryotes. DCAF13 contains three WD40 domains and is hypothesized to have roles in both rRNA processing and in ubiquitination of proteins. Analysis of DCAF13 with the p.(Asp455Asn) variant predicted that the amino acid change is deleterious and affects a ß-hairpin turn, within a WD40 domain of the protein which may decrease protein stability. Previously, a heterozygous variant of DCAF13 NM_015420.6, c.20 G > C:p.(Trp7Ser) with or without a heterozygous missense variant in CCN3, was suggested to cause inherited cortical myoclonic tremor with epilepsy. In addition, a heterozygous DCAF13 variant has been associated with autism spectrum disorder. Our study indicates a potential role of biallelic DCAF13 variants in neuromuscular disorders. Screening of additional patients with similar phenotype may broaden the allelic and phenotypic spectrum due to DCAF13 variants.

Novel cis compound heterozygous variants in MYO6 causes early onset of non-syndromic hearing loss in a Chinese family.

Background: Mutations in the MYO6 gene have been associated with both autosomal dominant non-syndromic hearing loss (ADNSHL) and autosomal recessive non-syndromic hearing loss (ARNSHL), with a cumulative identification of 125 pathogenic variants. To investigate the underlying genetic factor within a Chinese family affected with heriditary hearing loss, prompted the utilization of high-throughput sequencing. Method: A detailed clinical investigation was performed. Genetic testing was performed by using target panel sequencing, and Sanger sequencing. Targeted sequencing identified the variants and Sanger sequencing was employed to validate segregation of the identified variants within family. Additionally, bioinformatics analysis was performed to strengthen our findings. Results: Clinical investigation revealed the family members were affected by progressive and sensorineural hearing loss with an onset around 8-10 years old. Furthermore, genetic testing identified novel MYO6 variants, c.[2377T>G; 2382G>T] p.[Trp793Gly; Lys794Asn], positioned in a cis pattern, as plausible pathogenic contributors to early-onset hearing loss characterized by a severe and progressive course. Moreover, bioinformatics analysis showd disruptin in hydrogen bonding of mutant amino acids with interactive amino acids. Conclusion: Our research uncovered a relationship between mutations in the MYO6 gene and non-syndromic hearing loss. We identified two variants, c.[2377T>G; 2382G>T] p.[Trp793Gly; Lys794Asn] in MYO6 as strong candidates responsible for the observed progressive hereditary hearing loss. This study not only adds to our knowledge about hearing problems related to MYO6 but also reveals the presence of monogenic compound heterozygosity. Our study will provide a new sight for genetic diagnosis in such patients and their management for future use.

Mutation in XPO5 causes adult-onset autosomal dominant familial focal segmental glomerulosclerosis.

BACKGROUND: Focal and segmental glomerulosclerosis (FSGS) is a histological pathology that characterizes a wide spectrum of diseases. Many genes associated with FSGS have been studied previously, but there are still some FSGS families reported in the literature without the identification of known gene mutations. The aim of this study was to investigate the new genetic cause of adult-onset FSGS. METHODS: This study included 40 FSGS families, 77 sporadic FSGS cases, 157 non-FSGS chronic kidney disease (CKD) families and 195 healthy controls for analyses. Whole-exome sequencing (WES) and Sanger sequencing were performed on probands and family members of all recruited families and sporadic FSGS cases. RESULTS: Using WES, we have identified a novel heterozygous missense variant (c.T1655C:p.V552A) in exportin 5 gene (XPO5) in two families (FS-133 and CKD-05) affected with FSGS and CKD. Sanger sequencing has confirmed the co-segregation of this identified variant in an autosomal dominant pattern within two families, while this variant was absent in healthy controls. Furthermore, the identified mutation was absent in 195 ethnically matched healthy controls by Sanger sequencing. Subsequently, in silico analysis demonstrated that the identified variant was highly conservative in evolution and likely to be pathogenic. CONCLUSIONS: Our study reports an adult-onset autosomal dominant inheritance of the XPO5 variant in familial FSGS for the first time. Our study expanded the understanding of the genotypic, phenotypic and ethnical spectrum of mutation in this gene.

Association between XRCC3 p.Thr241Met polymorphism and risk of glioma: A systematic review and meta-analysis.

BACKGROUND: The XRCC3 p.Thr241Met (rs861539) polymorphism has been extensively studied for its association with glioma risk, but results remain conflicting. Therefore, we performed a systematic review and meta-analysis to resolve this inconsistency. METHODS: Studies published up to June 10, 2022, were searched in PubMed, Web of Science, Scopus, VIP, Wanfang, and China National Knowledge Infrastructure databases and screened for eligibility. Then, the combined odds ratio (OR) of the included studies was estimated based on five genetic models, i.e., homozygous (Met/Met vs. Thr/Thr), heterozygous (Thr/Met vs. Thr/Thr), dominant (Thr/Met + Met/Met vs. Thr/Thr), recessive (Met/Met vs. Thr/Thr + Thr/Met) and allele (Met vs. Thr). The study protocol was preregistered at PROSPERO (registration number: CRD42021235704). RESULTS: Overall, our meta-analysis of 14 eligible studies involving 12,905 subjects showed that the p.Thr241Met polymorphism was significantly associated with increased glioma risk in both homozygous and recessive models (homozygous, OR = 1.381, 95% CI = 1.081-1.764, P = 0.010; recessive, OR = 1.305, 95% CI = 1.140-1.493, P<0.001). Subgroup analyses by ethnicity also revealed a statistically significant association under the two aforementioned genetic models, but only in the Asian population and not in Caucasians (P>0.05). CONCLUSION: We demonstrated that the XRCC3 p.Thr241Met polymorphism is associated with an increased risk of glioma only in the homozygous and recessive models.

A Validation Study Comparing Risk Prediction Models of IgA Nephropathy.

We aimed to validate three IgAN risk models proposed by an international collaborative study and another CKD risk model generated by an extended CKD cohort with our multicenter Chinese IgAN cohort. Biopsy-proven IgAN patients with an eGFR ≥15 ml/min/1.73 m2 at baseline and a minimum follow-up of 6 months were enrolled. The primary outcomes were a composite outcome (50% decline in eGFR or ESRD) and ESRD. The performance of those models was assessed using discrimination, calibration, and reclassification. A total of 2,300 eligible cases were enrolled. Of them, 288 (12.5%) patients reached composite outcome and 214 (9.3%) patients reached ESRD during a median follow-up period of 30 months. Using the composite outcome for analysis, the Clinical, Limited, Full, and CKD models had relatively good performance with similar C statistics (0.81, 0.81, 0.82, and 0.82, respectively). While using ESRD as the end point, the four prediction models had better performance (all C statistics > 0.9). Furthermore, subgroup analysis showed that the models containing clinical and pathological variables (Full model and Limited model) had better discriminatory abilities than the models including only clinical indicators (Clinical model and CKD model) in low-risk patients characterized by higher baseline eGFR (≥60 ml/min/1.73 m2). In conclusion, we validated recently reported IgAN and CKD risk models in our Chinese IgAN cohort. Compared to pure clinical models, adding pathological variables will increase performance in predicting ESRD in low-risk IgAN patients with baseline eGFR ≥60 ml/min/1.73 m2.

Hypophosphatemia is an independent risk factor for AKI among hospitalized patients with COVID-19 infection.

BACKGROUND: This study sought to investigate incidence and risk factors for acute kidney injury (AKI) in hospitalized COVID-19. METHODS: In this retrospective study, we enrolled 823 COVID-19 patients with at least two evaluations of renal function during hospitalization from four hospitals in Wuhan, China between February 2020 and April 2020. Clinical and laboratory parameters at the time of admission and follow-up data were recorded. Systemic renal tubular dysfunction was evaluated via 24-h urine collections in a subgroup of 55 patients. RESULTS: In total, 823 patients were enrolled (50.5% male) with a mean age of 60.9 ± 14.9 years. AKI occurred in 38 (40.9%) ICU cases but only 6 (0.8%) non-ICU cases. Using forward stepwise Cox regression analysis, we found eight independent risk factors for AKI including decreased platelet level, lower albumin level, lower phosphorus level, higher level of lactate dehydrogenase (LDH), procalcitonin, C-reactive protein (CRP), urea, and prothrombin time (PT) on admission. For every 0.1 mmol/L decreases in serum phosphorus level, patients had a 1.34-fold (95% CI 1.14-1.58) increased risk of AKI. Patients with hypophosphatemia were likely to be older and with lower lymphocyte count, lower serum albumin level, lower uric acid, higher LDH, and higher CRP. Furthermore, serum phosphorus level was positively correlated with phosphate tubular maximum per volume of filtrate (TmP/GFR) (Pearson r = 0.66, p < .001) in subgroup analysis, indicating renal phosphate loss via proximal renal tubular dysfunction. CONCLUSION: The AKI incidence was very low in non-ICU patients as compared to ICU patients. Hypophosphatemia is an independent risk factor for AKI in patients hospitalized for COVID-19 infection.

A validation study of UCSD-Mayo risk score in predicting hospital-acquired acute kidney injury in COVID-19 patients.

INTRODUCTION: Acute kidney injury (AKI) in coronavirus disease 2019 (COVID-19) patients is associated with poor prognosis. Early prediction and intervention of AKI are vital for improving clinical outcome of COVID-19 patients. As lack of tools for early AKI detection in COVID-19 patients, this study aimed to validate the USCD-Mayo risk score in predicting hospital-acquired AKI in an extended multi-center COVID-19 cohort. METHODS: Five hundred seventy-two COVID-19 patients from Wuhan Tongji Hospital Guanggu Branch, Wuhan Leishenshan Hospital, and Wuhan No. Ninth Hospital was enrolled for this study. Patients who developed AKI or reached an outcome of recovery or death during the study period were included. Predictors were evaluated according to data extracted from medical records. RESULTS: Of all patients, a total of 44 (8%) developed AKI. The UCSD-Mayo risk score achieved excellent discrimination in predicting AKI with the C-statistic of 0.88 (95%CI: 0.84-0.91). Next, we determined the UCSD-Mayo risk score had good overall performance (Nagelkerke R2 = 0.32) and calibration in our cohort. Further analysis showed that the UCSD-Mayo risk score performed well in subgroups defined by gender, age, and several chronic comorbidities. However, the discrimination of the UCSD-Mayo risk score in ICU patients and patients with mechanical ventilation was not good which might be resulted from different risk factors of these patients. CONCLUSIONS: We validated the performance of UCSD-Mayo risk score in predicting hospital-acquired AKI in COVID-19 patients was excellent except for patients from ICU or patients with mechanical ventilation.

Next-Generation Sequencing Identifies Pathogenic Variants in HGF, POU3F4, TECTA, and MYO7A in Consanguineous Pakistani Deaf Families.

Background: Approximately 70% of congenital deafness is attributable to genetic causes. Incidence of congenital deafness is known to be higher in families with consanguineous marriage. In this study, we investigated the genetic causes in three consanguineous Pakistani families segregating with prelingual, severe-to-profound deafness. Results: Through targeted next-generation sequencing of 414 genes known to be associated with deafness, homozygous variants c.536del (p. Leu180Serfs∗20) in TECTA, c.3719 G>A (p. Arg1240Gln) in MYO7A, and c.482+1986_1988del in HGF were identified as the pathogenic causes of enrolled families. Interestingly, in one large consanguineous family, an additional c.706G>A (p. Glu236Lys) variant in the X-linked POU3F4 gene was also identified in multiple affected family members causing deafness. Genotype-phenotype cosegregation was confirmed in all participating family members by Sanger sequencing. Conclusions: Our results showed that the genetic causes of deafness are highly heterogeneous. Even within a single family, the affected members with apparently indistinguishable clinical phenotypes may have different pathogenic variants.

Whole exome sequencing identified a novel missense alteration in CC2D2A causing Joubert syndrome 9 in a Pakhtun family.

BACKGROUND: Joubert syndrome (JBTS) is a heterogenous disorder characterized by intellectual disability, developmental delays, molar tooth sign in brain imaging, hypotonia, ocular motor apraxia and overlapping features of ciliopathies. There are 36 clinical subtypes of JBTS, with an equal number of genes known so far for this phenotype. METHODS: Whole exome sequencing (WES) and Sanger sequencing were performed for the molecular diagnosis of a Pakhtun family affected with Joubert syndrome type 9 (JBTS9). RESULTS: A novel homozygous missense variant (c.4417C>G; Pro1473Ala) in exon 34 was identified in coiled-coil and C2 domains-containing the protein 2A (CC2D2A; NM_001080522) gene. The variant co-segregated in autosomal recessive fashion within the family and was not found in 200 ethnically matched unaffected individuals. In silico analyses supported the pathogenic effect of the altered CC2D2A protein. CONCLUSIONS: To the best of our knowledge, this is the first report of CC2D2A alteration co-segragating with a JBTS9 phenotype in a Pakhtun family from Pakistan. Our findings broaden the pathogenic spectrum of JBTS9, adding a novel variant to CC2D2A variation pool. WES analysis is a successful molecular diagnostic tool for rare genetic disorders, especially in those populations where the marriage of cousins is more frequent. Efficient and accurate genetic testing and counselling of the affected families are helpful for patient management and for reducing the disease burden in future generations.

Au@Ag core-shell nanoparticles for microRNA-21 determination based on duplex-specific nuclease signal amplification and surface-enhanced Raman scattering.

A novel surface-enhanced Raman scattering (SERS) analysis strategy has been designed combining Au@DTNB@Ag core-shell nanoparticles (DTNB attachment on gold nanoparticles, then encapsulated in Ag shell nanoparticles named as ADANPs) and duplex-specific nuclease signal amplification (DSNSA) platform. Firstly, ADANPs and magnetic substrate of Fe3O4 nanoparticles were covalently attached to the 3'- and 5'- end of capture probe (CP) targeting miRNA-21. Upon the addition of target miRNA-21, these heteroduplexes were specifically cleaved by DSN and resulted in ADANPs that were released from the surface of Fe3O4 nanoparticles (Fe3O4 NPs). At the same time, miRNA-21 remained intact and can rehybridize another DNA probe to trigger the signal-amplifying reaction. Based on this principle, the developed SERS method exhibited good linearity in the range 0 to 1 nM for miRNA-21 with a limit of detection (LOD) of 0.084 fM and has an ability to differentiate even a single-base mismatched sequence on the target sequence or other miRNA sequence. The results provide a novel SERS method which can successfully been applied to the miRNA-21 detection in human serum. Graphical abstract a shows the synthesis of Fe3O4 NPs and the conjugation of Au@DTNB@Ag NPs (ADANPs) for the detection of miRNA-21, b shows the operating principle of DSN-assisted signal amplification strategy for miRNA detection based on Fe3O4@CP@ADA NPs.

A highly sensitive DNAzyme-based SERS biosensor for quantitative detection of lead ions in human serum.

Lead ions (Pb2+), one form of the toxic heavy metal, have drawn significant attention due to their harmful effects on human health and the environment. Although many analytical techniques have been developed over the past few decades, the development of a sensitive, selective, and rapid method to detect Pb2+ remains a challenge. In this work, we developed a sensitive surface-enhanced Raman scattering (SERS) biosensor for highly sensitive detection of Pb2+ by using DNAzyme-modified Fe3O4@Au@Ag nanoparticles (Fe3O4@Au@Ag NPs). Firstly, the thiolated 5'-Cy3 DNA probe was modified on the surface of Fe3O4@Au@Ag NPs, which hybridized with the Pb2+-specific DNAzyme to form a SERS biosensor, and the Cy3 labels were used to detect Pb2+. In the presence of Pb2+, the DNAzyme cleaves the Cy3-labeled DNA probe, leading to the release of Cy3-labeled DNA probe from the Fe3O4@Au@Ag NPs. Therefore, the Raman intensity of the Cy3 labels decreases. The proposed biosensor exhibited excellent linearity in the range from 0.01 to 1.0 nM, with a limit of detection for Pb2+ of 5 pM. It features superior selectivity to Pb2+ over other interfering metal ions and good application in the determination of Pb2+ in tap water and human serum samples. The SERS biosensor provides a novel' simple and sensitive method for detection of Pb2+ and sheds new light on the design and synthesis of analogous SERS biosensors for the detection of other heavy metal ions.

Clinical and genetic analysis of lipoprotein glomerulopathy patients caused by APOE mutations.

BACKGROUND: Lipoprotein glomerulopathy (LPG) is a rare kidney disease caused by APOE mutations. The aim of this study was to correlate the genetic and clinical features of LPG. METHODS: Totally eight LPG patients were recruited in this study and Sanger sequencing of APOE was performed for all available family members. Clinical and histological features were analyzed. A literature review of LPG was also conducted. RESULTS: Genetic analysis revealed five patients with APOE-Kyoto, two with APOE-Osaka/Kurashiki, and one with APOE-Chicago mutations. LPG patients with urine protein reduced more than 50% had a slower decrease in renal function than those with less urine protein reduction (estimated glomerular filtration rate reduction rate -5.0 ± 0.8 vs. 1.5 ± 0.7 ml/min per 1.73 m2 ⋅month-1 , p = .03). We then enrolled 95 LPG patients from previous studies and this study. LPG patients had higher blood pressure (mean arterial pressure: 109.4 ± 19.4 vs. 94.4 ± 11.1 mmHg, p < .001) than the control group. Interestingly, patients with APOE mutations in the LDL receptor binding region had higher serum apolipoprotein E (apoE) levels [ln(apoE): 2.7 ± 0.4 vs. 2.0 ± 0.5 mg/dl, p < .001] in comparison to other domains. CONCLUSION: Here, we report for the first time APOE-Osaka/Kurashiki and APOE-Chicago mutations in the Chinese population. LPG was associated with higher blood pressure and serum apoE levels were higher in patients with mutations in LDL receptor binding region. In addition, the findings further indicated that treatment of proteinuria might slow down renal function progression in these patients.

A TOP6BL mutation abolishes meiotic DNA double-strand break formation and causes human infertility.

Meiosis is pivotal for sexual reproduction and fertility. Meiotic programmed DNA double-strand breaks (DSBs) initiate homologous recombination, ensuring faithful chromosome segregation and generation of gametes. However, few studies have focused on meiotic DSB formation in human reproduction. Here, we report four infertile siblings born to a consanguineous marriage, with three brothers suffering from non-obstructive azoospermia and one sister suffering from unexplained infertility with normal menstrual cycles and normal ovary sizes with follicular activity. An autosomal recessive mutation in TOP6BL was found co-segregating with infertility in this family. Investigation of one male patient revealed failure in programmed meiotic DSB formation and meiotic arrest prior to pachytene stage of prophase I. Mouse models carrying similar mutations to that in patients recapitulated the spermatogenic abnormalities of the patient. Pathogenicity of the mutation in the female patient was supported by observations in mice that meiotic programmed DSBs failed to form in mutant oocytes and oocyte maturation failure due to absence of meiotic recombination. Our study thus illustrates the phenotypical characteristics and the genotype-phenotype correlations of meiotic DSB formation failure in humans.

A DNAH17 missense variant causes flagella destabilization and asthenozoospermia.

Asthenozoospermia is a common cause of male infertility, but its etiology remains incompletely understood. We recruited three Pakistani infertile brothers, born to first-cousin parents, displaying idiopathic asthenozoospermia but no ciliary-related symptoms. Whole-exome sequencing identified a missense variant (c.G5408A, p.C1803Y) in DNAH17, a functionally uncharacterized gene, recessively cosegregating with asthenozoospermia in the family. DNAH17, specifically expressed in testes, was localized to sperm flagella, and the mutation did not alter its localization. However, spermatozoa of all three patients showed higher frequencies of microtubule doublet(s) 4-7 missing at principal piece and end piece than in controls. Mice carrying a homozygous mutation (Dnah17M/M) equivalent to that in patients recapitulated the defects in patients' sperm tails. Further examinations revealed that the doublets 4-7 were destabilized largely due to the storage of sperm in epididymis. Altogether, we first report that a homozygous DNAH17 missense variant specifically induces doublets 4-7 destabilization and consequently causes asthenozoospermia, providing a novel marker for genetic counseling and diagnosis of male infertility.

The testis-specifically expressed Dpep3 is not essential for male fertility in mice.

More than 2300 genes have been reported to be involved in spermatogenesis but the functional roles of most genes in male fertility remain to be elucidated. In this study, we explored the function of dipeptidase 3 (Dpep3), a gene predicted to be testis-specific, in male fertility of mice. We showed that Dpep3 is evolutionarily conserved in human and mouse along with other eutherians. Its mRNA was exclusively detected in testicular tissue and expressed in testes from 7 days postpartum. To further explore its role in male fertility, we generated Dpep3 knockout mice (Dpep3-/-) using the CRISPR/Cas9 technology and found that the male Dpep3-/- mice are fertile despite a significant reduction in sperm count. Histology of testis and progression of meiotic prophase I showed no obvious difference between wild-type and Dpep3-/- mice. All these findings indicate that Dpep3 is not essential for male fertility in mice. These findings will help other researchers to avoid research duplication, save their time and resources to focus on the genes that are indispensable for male fertility.

Identification of CDKN2A variants in breast cancer patients in Pakistan.

The role of cyclin-dependent kinase inhibitor 2A gene (CDKN2A) variants in breast cancer is not well understood, here we investigated their possible effects on breast cancer in Pakistani women attending the NORI Hospital, Islamabad. Direct DNA sequencing of CDKN2A identified an already known polymorphism in the 3' UTR, c.*29G>C (rs11515), in 5.88% patients and two novel variants. One, a deep intronic substitution (c.458-554T>G) in 1.96% patients, is also detected as a compound heterozygous form along with c.*29G>C in 1.96% patients (c.[458-554T>G; *29G>C]). The other is a novel deletion (c.458-82delG) occurring as a compound variant with two other identified variants c.[458-554T>G; 458-82delG; *29G>C] in 1.96% patients. In silico pathogenicity prediction analyses did not predict pathogenic effects on breast cancer for these individual variants. We conclude that variations in CDKN2A are not the major genetic cause of breast cancer in the enrolled Pakistani patients.

Whole Exome Sequencing Revealed a Novel Nonsense Variant in the GNRHR Gene Causing Normosmic Hypogonadotropic Hypogonadism in a Pakistani Family.

BACKGROUND: Congenital hypogonadotropic hypogonadism (CHH) is a heterogeneous disorder characterized by delayed or loss of puberty and infertility due to functional deficiency in the hypothalamic gonadotropin-releasing hormone (GnRH). CHH can be classified into 2 subtypes on the basis of olfaction: Kallmann syndrome and normosmic CHH (nCHH). The spectrum of genetic variants causing CHH is continually expanding. Here, we recruited a consanguineous Pakistani family having 2 male and 2 female infertile patients diagnosed with idiopathic nCHH. AIMS: The aim of this study was to investigate the genetic cause of nCHH in the family. METHODS: Clinical and physical analyses were performed for the patients. Genetic analysis was carried out using whole exome and Sanger sequencing. RESULTS: Clinical and physical investigations confirmed low levels of gonadotropins and failure of secondary sexual development in the patients. Genetic analysis identified a novel nonsense mutation (chr4: g.68619942G>A, c.112C>T, p.Arg38*) in the gonadotropin-releasing hormone receptor gene (GNRHR) recessively co-segregating with nCHH in this family. All the patients are homozygous and their parents are heterozygous carriers, while normal siblings are heterozygous carriers or wild-type for this mutation, indicating that the identified mutation is pathogenic for nCHH in the family. CONCLUSION: We report the first homozygous nonsense mutation in the GNRHR gene (chr4: g. 68619942G>A, c.112C>T, p. Arg38*) that is associated with familial nCHH. Hence, our study displayed a good correlation of the genotype and phenotype of nCHH patients.