Clinical experience of next generation sequencing based expanded carrier screening in high-risk couples from a tertiary healthcare center in Pakistan.

INTRODUCTION: Carrier screening for genetic conditions has long been a part of preconception and prenatal care. While the use of expanded carrier screening (ECS) is widely common in HICs (high income countries), the clinical actionability of ECS in LMICs (low middle income countries) with high consanguineous unions is not well-understood. METHOD: Retrospective chart review of couples who presented to the Prenatal Genetics Clinic at Aga Khan University Hospital, between the period of June 2018 and November 2022. All the statistical analyses were performed using the statistical software STATA version 17.0. RESULTS: Of the 202 individuals tested, 166 (82%) were identified to be carriers of at least one gene associated with a monogenic condition. Out of the 302 genes tested, individuals were found to be carriers of conditions associated with 87 genes. Clinical actionability of ECS was established in a total of 45 (45%) high risk couples undergoing screening using ECS. CONCLUSION: We report the first clinical experience of using next generation sequencing based ECS in 101 high-risk couples seeking consultation in the Genetics Clinic, at a tertiary healthcare center in Pakistan, showing that over 80% high-risk individuals are carriers of at least one condition and 45% of couples receive an actionable result to making autonomous informed reproductive decisions in future pregnancies.

Evaluation of the clinical, biochemical, and genetic presentation of neonatal and adult-onset 5,10-methylene tetrahydrofolate reductase (MTHFR) deficiency in patients from Pakistan.

OBJECTIVES: To study the biochemical, clinical and molecular characteristics of 5,10- methylenetetrahydrofolate reductase (MTHFR) deficiency in Pakistani patients from a single center. METHODS: Medical charts, urine organic acid chromatograms, plasma methionine and Hcys levels, and molecular testing results of MTHFR gene of patients presenting at the Biochemical Genetics Clinic, AKUH from 2016 to 2022 were reviewed. RESULTS: Neonatal MTHFR deficiency was found in five patients. The median (IQR) age of symptom onset and diagnosis were 18 (8.5-22) and 26 (16.5-31) days. The median lag between symptom onset and diagnosis was 8 (4.5-12.5) days. The median age of treatment initiation and duration of treatment were 26 (16.5-49) and 32 (25.5-54) days. The most common clinical features were lethargy, poor feeding, and seizures. The MTHFR gene sequencing revealed homozygous variants p.K510K, p.R567*, and p.R157W. Renal insufficiency manifesting as elevated serum creatinine and responding to betaine therapy was noted in one patient. This has not been previously reported in neonatal MTHFR deficiency and may reflect engagement of alternate pathways of remethylation. Adult onset MTHFR deficiency was found in six patients, with a heterogeneous neurological presentation. The median lag between symptoms onset and diagnosis was 7 (3-11) years. MTHFR gene sequencing revealed homozygous variant p.A195V in five patients from one family and p.G261V in the other. Two of the five reported variants are novel that include p.R157W and p.G261V. CONCLUSIONS: Eleven patients of this rare disorder from a single center indicate the need for clinical awareness and appropriate biochemical evaluation to ensure optimal outcomes.

Experience in prenatal genetic testing and reproductive decision-making for monogenic disorders from a single tertiary care genetics clinic in a low-middle income country.

OBJECTIVES: Explore health-care seeking behaviour among couples with pregnancies at-risk of monogenic disorders and compare time duration for obtaining Prenatal Genetic Test (PGT) results based on (i) amniocentesis and Chorionic Villus Sampling (CVS) (ii) in-house testing and out-sourced testing. Report the spectrum of monogenic disorders in our cohort. METHODS: Medical records of women consulting prenatal genetic counselling clinic at Aga Khan University Hospital, Karachi from December-2015 to March-2021 with history of miscarriage or a monogenic disorder in previous children were reviewed. RESULTS: Forty-three pregnancies in 40 couples were evaluated, 37(93%) were consanguineous. Twenty-five (63%) couples consulted before and 15(37%) after conception. Thirty-one (71%) pregnancies underwent CVS at the mean gestational age of 13-weeks and 6-days ± 1-week and 3-days and amniocentesis at 16-weeks and 2-days ± 1-week and 4-days. PGT for 30 (70%) pregnancies was outsourced. The mean number of days for in-house PGT was 16.92 ± 7.80 days whereas for outsourced was 25.45 ± 7.7 days. Mean duration from procedure to PGT result was 20.55 days after CVS compared to 28.75 days after amniocentesis. Eight (18%) fetuses were homozygous for disease-causing variant for whom couples opted for termination of pregnancy (TOP). Twenty-six monogenetic disorders were identified in 40 families. CONCLUSION: Proactive health-care seeking behaviour and TOP acceptance is present amongst couples who have experienced a genetic disorder.

The spectrum of hereditary neuromuscular disorders in the Pakistani population.

Hereditary neuromuscular disorders (NMDs) are a broad group of clinically heterogeneous disorders with varying inheritance patterns, that are associated with over 500 implicated genes. In the context of a highly consanguineous Pakistani population, we expect that autosomal recessive NMDs may have a higher prevalence compared with patients of European descent. This is the first study to offer a detailed description of the spectrum of genes causing hereditary NMDs in the Pakistani population using NGS testing. To study the clinical and genetic profiles of patients presenting for evaluation of a hereditary neuromuscular disorder. This is a retrospective chart review of patients seen in the Neuromuscular Disorders Clinic and referred to the Genetics Clinic with a suspected hereditary neuromuscular disorder, between 2016 and 2020 at the Aga Khan University Hospital, Karachi and Mukhtiar A. Sheikh Hospital, Multan, Pakistan. The genetic testing for these patients included NGS-based single gene sequencing, NGS-based multi-gene panel and whole exome sequencing. In a total of 112 patients studied, 35 (31.3%) were female. The mean age of onset in all patients was 14.6 years (SD ±12.1 years), with the average age at presentation to the clinic of 22.4 years (SD ±14.10 years). Forty-seven (41.9%) patients had a positive genetic test result, 53 (47.3%) had one or more variants of uncertain significance (VUS), and 12 (10.7%) had a negative result. Upon further genotype-phenotype correlation and family segregation analysis, the diagnostic yield improved, with 59 (52.7%) patients reaching a diagnosis of a hereditary NMD. We also report probable founder variants in COL6A2, FKTN, GNE, and SGCB, previously reported in populations that have possible shared ancestry with the Pakistani population. Our findings reemphasizes that the rate of VUSs can be reduced by clinical correlation and family segregation studies.

Prophylactic Risk-Reducing Mastectomy (PRRM): A Set Practice or Catch-22 Situation in LMIC. A Single-Centre Prospective Cohort Study.

Background Pakistan's hereditary breast cancer has a higher-than-average prevalence. Our acceptability of prophylactic risk-reducing mastectomy (PRRM) still needs to be determined, and genetic testing still needs to be offered to all eligible. The aim is to determine the number of women presenting to our centre who availed of PRRM after positive genetic tests and the main reasons restraining them from considering PRRM.Materials and methods This study is a single-centre, prospective cohort. We collected data from 2017 to 2022 on BRCA1/2 and other (P/LP) gene-positive patients. Continuous variables are presented as means (±SD) and categorical variables in percentages, with a significant P-value of ≤ 0.05.Results Out of 477 tested individuals, only 95(20.12%) had a positive result. BRCA1/2 was positive in 70 cases, while P/LP variants were in 24 cases. Only 32.6% of eligible families underwent genetic testing, with 54.8% positivity. Altogether, 92.6% of patients had BRCA1/2-related cancers. Only 25/95(26.3%) individuals availed of PRRM, the majority had contralateral risk-reducing mastectomy 68% with a 20% reconstruction rate. The main reasons to decline PRRM were false belief of not having any disease 57.44%, followed by family/spouse pressure 51%, body appearance/societal perception, fear of complications/quality of life and financial constraints.Conclusion Genetic testing and its implications are still a grey area for LMICs, primarily due to the scarcity of centres offering genetic testing to eligible populations, followed by prevalent perceptions about prophylactic surgeries among the masses. Addressing relevant issues in LMICs is the need of time.

Utility of genetic testing in pediatric epilepsy: Experience from a low to middle-income country.

Monogenic epilepsies are a significant etiology of pediatric epilepsy. These are now more easily identified due to advances in genetic testing. However, the utility of genetic testing in low to middle-income countries (LMICs) has not been fully explored. A retrospective review was carried out in Karachi, Pakistan. Patients with symptoms suggestive of genetic epilepsy underwent next-generation sequencing (NGS). Seventy-seven patients were tested, of which 27 % (n = 21) initially had pathogenic (P) or likely pathogenic (LP) results. This increased to 32 % (n = 25) after clinical reclassification of some variants of uncertain significance (VUSs) based on American College of Medical Genetics and Genomics (ACMG) guidelines. Initially, 6 % of patients (n = 5) had no P/LP or VUS, and 66 % (n = 51) had at least one VUS. After variant resolution and reclassification, results were negative for 25% (n = 19) and 43% (n = 33) had VUSs. Genetic testing was positive in one-third of our population. The proportion of P/LP variants found in SCN1A is higher than that found in other populations, and we report two novel variants in SCN1A. The yield of genetic testing in our population is comparable to that found in North America. Initially, a higher proportion of our population had inconclusive results, indicating the need for better characterization of the South Asian genotype.

Novel Homozygous TTI2 Variant Causing Autosomal Recessive Syndromic Intellectual Disability and Primary Microcephaly from Pakistan: A Case Report (Exome Report).

We describe a male patient with a novel TTI2 variant, which has not been previously associated with a human phenotype. His features include intellectual disability, primary microcephaly, delayed psychomotor development, speech delay, short stature, dysmorphic facial features, esotropia, kyphoscoliosis, and behavior abnormalities (Figure). Next generation sequencing revealed autosomal recessive TTI2 variant with uncertain significance, denoted as c.21_22insAAGCGCTCTG (p.Glu8Lysfs × 12). TTI2 encodes a regulator of DNA damage response and helps maintain steady levels of the PIKK family of protein kinases. No disease-causing variants in other genes potentially linked to his clinical presentation were identified. We report a novel loss-of-function homozygous variant in TTI2 that leads to syndromic intellectual disability and primary microcephaly.

Spectrum of germline pathogenic variants using a targeted next generation sequencing panel and genotype-phenotype correlations in patients with suspected hereditary breast cancer at an academic medical centre in Pakistan.

BACKGROUND: Breast cancer is the most common malignancy in women, affecting over 1.5 million women every year, which accounts for the highest number of cancer-related deaths in women globally. Hereditary breast cancer (HBC), an important subset of breast cancer, accounts for 5-10% of total cases. However, in Low Middle-Income Countries (LMICs), the population-specific risk of HBC in different ethnicities and the correlation with certain clinical characteristics remain unexplored. METHODS: Retrospective chart review of patients who visited the HBC clinic and proceeded with multi-gene panel testing from May 2017 to April 2020. Descriptive and inferential statistics were used to analyze clinical characteristics of patients. Fisher's exact, Pearson's chi-squared tests and Logistic regression analysis were used for categorical variables and Wilcoxon rank-sum test were used for quantitative variables. For comparison between two independent groups, Mann-Whitney test was performed. Results were considered significant at a p value of < 0.05. RESULTS: Out of 273 patients, 22% tested positive, 37% had a VUS and 41% had a negative genetic test result. Fifty-five percent of the positive patients had pathogenic variants in either BRCA1 or BRCA2, while the remaining positive results were attributed to other genes. Patients with a positive result had a younger age at diagnosis compared to those having a VUS and a negative result; median age 37.5 years, IQR (Interquartile range) (31.5-48). Additionally, patients with triple negative breast cancer (TNBC) were almost 3 times more likely to have a positive result (OR = 2.79, CI = 1.42-5.48 p = 0.003). Of all patients with positive results, 25% of patients had a negative family history of breast and/or related cancers. CONCLUSIONS: In our HBC clinic, we observed that our rate of positive results is comparable, yet at the higher end of the range which is reported in other populations. The importance of expanded, multi-gene panel testing is highlighted by the fact that almost half of the patients had pathogenic or likely pathogenic variants in genes other than BRCA1/2, and that our test positivity rate would have only been 12.8% if only BRCA1/2 testing was done. As the database expands and protocol-driven referrals are made across the country, our insight about the genetic architecture of HBC in our population will continue to increase.

Challenges and opportunities in the establishment of a hereditary breast cancer clinic at an academic medical center in a low-middle income country.

It is now standard of care to offer genetic testing to patients at risk of hereditary breast cancer and make management decisions based on these results. Although great strides have been made in ensuring access to genetic testing and genetic counseling by establishing hereditary breast cancer clinics in well-resourced countries, these are essentially non-existent in low-middle income countries like Pakistan. We established a hereditary breast cancer clinic involving a multidisciplinary team, including a medical geneticist and a genetic counselor. Our efforts were based on consensus guidelines and included educating medical providers about the importance of genetic testing in breast cancer care and the mandatory presence of a genetics team member at the weekly Breast Tumor Board meeting. This resulted in an increase in the number of referrals of breast cancer patients for genetic testing. In this report, we describe the challenges we faced in setting up such a system in Pakistan and the measures to overcome them. There is a need to establish such hereditary breast cancer clinics, which can also be replicated at other centers in low-resource settings, to improve standardized assessment and management of the patients with hereditary breast cancer according to consensus guidelines.

Clinical, pathological and molecular spectrum of patients with glycogen storage diseases in Pakistan.

OBJECTIVES: Evaluation of clinical, biochemical and molecular analysis of Pakistani patients with hepatic GSDs. METHODS: Medical charts, biochemical, histopathological and molecular results of patients with hepatic GSD were reviewed. RESULTS: Out of 55 GSD patients, 41 (74.5%) were males and 14 (25.5%) were females with consanguinity in 50 (91%) patients. The median age of initial symptoms, clinic diagnosis and molecular diagnosis were 450 (IQR: 270-960), 1,095 (IQR: 510-1,825) and 1717 (IQR: 796-3,011) days, respectively. Molecular analysis and enzyme activity was available for 33 (60%) and two patients, respectively. GSD III (n=9) was most prevalent followed by GSD Ib (n=7), GSD IXc (n=6), GSD VI (n=4), GSD Ia (n=3), GSD XI (n=3), GSD IXb (n=2) and GSD IXa (n=1). In patients (n=33) who underwent molecular analysis; 19 different variants in eight genes associated with GSD were identified. We also report five novel variants, two in SLC37A4, one in AGL and two in PYGL contributing to the diagnosis of GSD Ib, GSD III and GSD VI, respectively. CONCLUSIONS: Fifty-five patients of GSDs in 26 families from a single care provider indicate a relatively high frequency of GSD in Pakistan, with multiple unrelated families harboring identical disease-causing variants, on molecular analysis, including two known pathogenic variants in SLC37A4 and PHKG2, and a novel variant in AGL.

PIGG variant pathogenicity assessment reveals characteristic features within 19 families.

PURPOSE: Phosphatidylinositol Glycan Anchor Biosynthesis, class G (PIGG) is an ethanolamine phosphate transferase catalyzing the modification of glycosylphosphatidylinositol (GPI). GPI serves as an anchor on the cell membrane for surface proteins called GPI-anchored proteins (GPI-APs). Pathogenic variants in genes involved in the biosynthesis of GPI cause inherited GPI deficiency (IGD), which still needs to be further characterized. METHODS: We describe 22 individuals from 19 unrelated families with biallelic variants in PIGG. We analyzed GPI-AP surface levels on granulocytes and fibroblasts for three and two individuals, respectively. We demonstrated enzymatic activity defects for PIGG variants in vitro in a PIGG/PIGO double knockout system. RESULTS: Phenotypic analysis of reported individuals reveals shared PIGG deficiency-associated features. All tested GPI-APs were unchanged on granulocytes whereas CD73 level in fibroblasts was decreased. In addition to classic IGD symptoms such as hypotonia, intellectual disability/developmental delay (ID/DD), and seizures, individuals with PIGG variants of null or severely decreased activity showed cerebellar atrophy, various neurological manifestations, and mitochondrial dysfunction, a feature increasingly recognized in IGDs. Individuals with mildly decreased activity showed autism spectrum disorder. CONCLUSION: This in vitro system is a useful method to validate the pathogenicity of variants in PIGG and to study PIGG physiological functions.

Assessing Health-Related Quality of Life, Morbidity, and Survival Status for Individuals With Down Syndrome in Pakistan (DS-Pak): Protocol for a Web-Based Collaborative Registry.

BACKGROUND: Down syndrome is the most common chromosomal disorder, with a global incidence of 1 in 700 live births. However, the true prevalence, associated morbidities, and health-related quality of life (HRQOL) of these individuals and their families are not well documented, especially in low- and middle-income countries such as Pakistan. Disease-specific documentation in the form of a collaborative registry is required to better understand this condition and the associated health outcomes. This protocol paper describes the aims and processes for developing the first comprehensive, web-based collaborative registry for Down syndrome in a Pakistani cohort. OBJECTIVE: This study aims to assess the HRQOL, long-term survival, and morbidity of individuals with Down syndrome by using a web-based collaborative registry. METHODS: The registry data collection will be conducted at the Aga Khan University Hospital and at the Karachi Down Syndrome Program. Data will be collected by in-person interviews or virtually via telephone or video interviews. Participants of any age and sex with Down syndrome (trisomy 21) will be recruited. After receiving informed consent and assent, a series of tablet-based questionnaires will be administered. The questionnaires aim to assess the sociodemographic background, clinical status, and HRQOL of the participants and their families. Data will be uploaded to a secure cloud server to allow for real-time access to participant responses by the clinicians to plan prompt interventions. Patient safety and confidentiality will be maintained by using multilayer encryption and unique coded patient identifiers. The collected data will be analyzed using IBM SPSS Statistics for Windows, Version 22.0 (IBM Corporation), with the mean and SD of continuous variables being reported. Categorical variables will be analyzed with their percentages being reported and with a P value cutoff of .05. Multivariate regression analysis will be conducted to identify predictors related to the HRQOL in patients with Down syndrome. Survival analysis will be reported using the Kaplan-Meier survival curves. RESULTS: The web-based questionnaire is currently being finalized before the commencement of pilot testing. This project has not received funding at the moment (ethical review committee approval reference ID: 2020-3582-11145). CONCLUSIONS: This registry will allow for a comprehensive understanding of Down syndrome in low- and middle-income countries. This can provide the opportunity for data-informed interventions, which are tailored to the specific needs of this patient population and their families. Although this web-based registry is a proof of concept, it has the potential to be expanded to national, regional, and international levels. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): PRR1-10.2196/24901.

Clinico-Pathological and Molecular Spectrum of Biotinidase Deficiency- Experience from a Lower Middle-Income Country.

BACKGROUND: The aim of this study was to evaluate the clinical, biochemical, and molecular analysis of Pakistani patients with biotinidase deficiency (BD). METHODS: Medical charts, urine organic acid (UOA) chromatograms, and biotinidase (BTD) enzyme activity of 113 suspected BD cases and BTD gene results of BTD enzyme deficient patients presenting at the Biochemical Genetics Clinic, AKUH from January 2010 to December 2019 were reviewed. Details were collected on a prestructured questionnaire. SPSS 22 was used for data analysis. RESULTS: BD was found in 33 (29.23%) cases, 28 being profound and 5 partial BD. The median age of BD diagnosis was 171 days (IQR: 81 - 1,022.75) and 300 days (IQR: 25 - 1,540) for the profound and partial BD, respectively. The median BTD levels in the partial BD and profound BD groups were 35 U (IQR: 25.5 - 62.5) and 15 U (IQR: 11 - 17), respectively. UOA analysis exhibited sensitivity, specificity, and agreement of 52.94%, 86.05%, and 76.67% with BTD enzyme activity. The BTD sequencing revealed seven recurrent homozygous single nucleotide variants (SNVs) and small indels. These variants include three frameshift, protein truncating variants and four missense variants. We report two novel protein truncating variants, c.929GinsA, p.S310fs*14 and c.394insA, p.T132Nfs*30 and one missense variant, c.416G>A, p.S139N that had not been reported in BD associated literature and clinical databases. CONCLUSIONS: Thirty-three cases of BD from a single center indicates a high frequency of BD in Pakistan. Late diagnosis emphasizes the need for increased clinical awareness and preferably screening for BD in this population.