Screening for cystic fibrosis: The importance of using the correct tools

Background: Cystic Fibrosis (CF) is a potentially lethal genetic disorder. The most frequent mutation worldwide in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene is designated as the Delta F508 mutation. This mutation was found in only 33% of Pakistani patients studied. Since the common Pakistani mutations remain to be identified, appropriate screening tools are required to identify disease. Sweat chloride determinations remain the gold standard for diagnosing CF. This study was done to emphasize the importance of using the correct tests.Methods: The study was conducted at the Aga Khan University Hospital. The CFTR delta F508 mutation was tested on blood samples from patients suspected with CF. Sweat chloride analysis using pilocarpine iontopharesis was done with a positive value of greater than 60 meq/L.Results: 57 pediatric samples were screened for the delta F508 mutation and were positive in only 10.6% of all patients tested. 12/57 (21%) had a preliminary sweat test. 6/12 (50%) of these patients had an abnormal sweat test and 3/6 patients with an abnormal sweat chloride (50%) had deltaF508 mutations-- 2/6 (33%) were homozygotes and 1 was a compound heterozygote. Since 79% did not have a sweat test, it was difficult to assess whether this subset of patients had cystic fibrosis with a CFTR mutation other than the delta F508 tested or no CF.Conclusion: Sweat chloride analysis is critical to distinguish CF from other causes of severe pulmonary and pancreatic insufficiencies and to define patients requiring further analysis

Evaluation of elevated alanine aminotransferase and hepatitis B virus DNA in healthy seronegative blood donors

Background: Serum alanine transaminase (ALT) has been used as a surrogate marker for detection of hepatitis B and C in blood donors in Pakistan since 1985. Since the introduction of more sensitive assays the value of ALT became questionable but it was still used with subsequent wastage of blood units with raised ALT. Finding: We conducted a study for a period of one year to evaluate the usefulness of ALT. During the study period, 25117 subjects donated blood. Eight hundred and seventy two donors (3.4%) were positive for one or more serological tests. ALT of all donors ranged from 0-1501 U/L (Mean±SD; 33.4±25.45U/L). The donors seronegative for all disease markers were 24245 (96.6%). Of these, 21164 (84.2%) donors had their ALT within reference range while 2874 (11.4%) and 207 (0.8%) of donors had minimal and markedly elevated results respectively. Six hundred and twenty one blood units (including red cells, platelets and plasma) were discarded based on elevated ALT results alone at a cost of $39,123. Two hundred seronegative blood donors with normal ALT, minimally and markedly elevated ALT levels, were selected randomly and evaluated for hepatitis B deoxynucleic acid by individual PCR. None of the donors was found to be reactive. Conclusion: This work did not support a positive association between hepatitis B virus nucleic acid and elevated ALT in healthy serologically negative blood donors

R990G Polymorphism of calcium sensing receptor gene Is associated with high Parathyroid Hormone levels in subjects with Vitamin D deficiency: a cross-sectional study

Single nucleotide polymorphisms (SNPs), R990G and A986S of the calcium sensing receptor (CaSR) gene, are shown to influence response of parathyroid hormone (PTH) in subjects with optimal vitamin D levels. This cross-sectional study was conducted in subjects with vitaminDdeficiency (VDD) to observe association’s betweenCaSR polymorphisms, plasma iPTH, and serumcalcium levels. Adult females ( = 140) with known VDD, intact parathyroid hormone (iPTH), and calcium levels were recruited for genotype analysis.The frequencies of the 986 alleles GG, GT, and TT were 68%, 25%, and 7%, respectively, whereas the frequencies of the 990 alleles AA, AG, and GG were 80%, 8.9%, and 11.1%, respectively.Thesubjects with GG genotype of R990G polymorphism had higher iPTH levels (148.65 versus 91.47 and 86.1 pg/mL for GG versus AA, AG, resp., = 0.008) and lower calcium levels (8.4 versus 9.04 and 9.07mg/dL for GG versus AA, AG, resp., = 0.002). No such association of A986S polymorphism with plasma iPTH or serum calcium levels was observed in the present study. Patients with VDD bearing the GG genotype of R990G SNPs are prone to have higher iPTH levels and lower calcium

Analysis of amniotic fluid specimens for common chromosome disorders using interphase fluorescence in situ hybridization

Objective: The aim of the study was to examine the usage of multi colour FISH technology as an adjunct to conventional cytogenetics for the prenatal diagnosis of aneuploidy in interphase nuclei from high risk pregnancies.Methods: Amniotic fluid samples were collected for interphase FISH analysis using DNA probes for chromosomes 13, 18, 21, X and Y. All the probes were directly labeled with fluorescent molecules. Fluorescent signals were observed under a microscope. A minimum of 100 nuclei with defined hybridization signals were counted for each probe. Results: Seventy-eight amniotic fluid samples were received for FISH analysis. The average age of mothers and their gestational ages were 33 years and 17.5 weeks respectively. Triple test screening was positive in 39.5% of the women followed by advanced maternal age and ultrasonographic abnormalities. Interphase FISH was performed on 76 specimens whereas 2 samples were rejected because of blood contamination. Aneuploidy was identified in 6 out of 76 specimens. Two cases of trisomy 21, two cases of trisomy 18 and one case of monosomy X were detected. In addition, one case showed 10% mosaicism for trisomy 21. Initially 4 (5.3%) samples were uninformative due to technical reasons but gave acceptable scoring signals when reanalyzed. Conclusion: This study has demonstrated that interphase FISH is a rapid and a reliable technique for the enumeration of chromosome number in uncultured amniocytes. Clinicians can use it for making early decisions necessary for the management of high risk pregnancies ultimately saving patients from anxiety and psychological stress (JPMA 57:189;2007

Frequency of ALK rearrangement by FISH testing and its correlation with ALK-IHC in adenocarcinoma of primary lung origin

Anaplastic lymphoma kinase (ALK) gene can be oncogenic either by forming fusion with other genes, amplification of the gene or by having mutations. ALK rearrangement can either be detected by standard “fluorescence in situ hybridization (FISH)” or “immunohistochemistry (IHC)”. Objective of this study was to record the prevalence of ALK rearrangement in adenocarcinoma of Primary Lung origin and compare it with ALK-IHC staining. Data of 64 patients of lung adenocarcinoma from 2015-2017 was analyzed. All of the FFPE biopsies were tested for EGFR (qPCR) followed by ALK rearrangement (by FISH and IHC) on EGFR negative samples. Out of 64 samples, 21.8% (14) showed EGFR mutations and 14% (7/50) were positive for ALK rearrangement when checked by FISH. In IHC testing for ALK (FISH positive) 8% (4/50) showed positivity. In conclusion ALK-FISH positive cases are higher than other studies likely due to the relatively small sample size. FISH testing was found to be more sensitive than IHC; one reason may be the low level of ALK. Our study warrants that currently FISH remains the gold standard for screening of ALK gene rearrangements

Effect of cytokine gene polymorphism on histological activity index, viral load and response to treatment in patients with chronic hepatitis C genotype 3

AIM: To investigate the association between cytokine gene polymorphism and disease status in chronic hepatitis C genotype 3 by liver biopsy, ALT, HCV RNA levels and response to treatment.Methods: Patients with chronic hepatitis C genotype 3 were analyzed for single nucleotide polymorphisms of interleukin (IL)-10, IL-1 beta, interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) by polymerase chain reaction using sequence-specific oligonucleotide primers. Liver biopsies were assessed by modified histological activity index (HAI) scoring system using a scale of 0-18 for grading the necro-inflammatory activity and 0-6 for staging the fibrosis. HCV RNA levels were determined by bDNA assay. The patients were treated with interferon alpha and ribavirin for 6 mo. Sustained virological response was assessed 6 mo after the completion of the treatment.Results: Out of the 40 patients analyzed, 26 were males. Mean age was 40.5+/-12.5 years (range 18-65 years). The frequencies of different dimorphic polymorphisms based on single nucleotide substitution were as follows: IL-10-1082 G/A 85%, A/A 12.5%, G/G 2.5%; IL-10-819 A/C 87.5%, C/C 10%, A/A 2.5%; IL-10-592 C/A 72.5%, C/C 27.5%; IL-1 C 90%, U 10%; IFN-874 T/A 50%, T/T 27.5%, A/A 22.5%; TNF-308 A/G 95%, G/G 5%; TGF-10 T/C 52.5%, C/C 35%, T/T 12.5%. The mean grades of necro-inflammatory activity of different genotypes of IL-10 at promoter site -1082 were A/A = 3.6, A/G = 5.0, and G/G = 10.0 and the difference was significant (P = 0.029). The difference in the stage of disease at a scale of 0-6 was A/A 0.8, A/G 2.3, and G/G 4.0 (P = 0.079). The difference in the HAI seemed to be related to the presence of allele -1082G. For IL-10 -819 genotypes, mean scores of fibrosis were A/A = 6.0, A/C = 2.2, and C/C = 1.0 (P = 0.020) though the inflammatory activity was not much different. No significant differences in HAI were noted among polymorphisms of other cytokines. Moreover, ALT and HCV RNA levels were not significantly different among different cytokine polymorphisms. There was a significant correlation of HAI and HCV RNA levels with the duration of disease. TGFbeta -10 genotype CC patients had a better end of treatment response than those with other genotypes (P = 0.020). Sustained virological response to the treatment was not influenced by the cytokine polymorphism. No effect of other factors like viral load, degree of fibrosis, gender, steatosis, was observed on sustained virological response in this population infected with genotype 3.CONCLUSION: There is no significant correlation between cytokine polymorphisms and HAI except for the polymorphisms of anti-inflammatory cytokine IL-10, which may influence hepatic inflammatory activity and fibrosis in patients with chronic hepatitis C genotype 3. Sustained virological response in this genotype does not seem to be influenced by cytokine gene polymorphisms

Assessment of WT1 expression as a marker of treatment outcome in karyotype normal acute myeloid leukemia patients in Pakistan

Currently, there is an effort to predict relapse by follow-up monitoring of MRD and subsequently to begin the treatment of the patients during their clinical and hematological remission prior to overt hematological relapse. Expression of WT1 in AM Lis known to be independently associated with significant inferior response to therapy and short survival outcome. Follow-up monitoring of WT1 gene expression during or after therapy would be a valuable predictive marker for early recurrence or relapse of AMLdisease. This pilot study evaluated newly diagnosed and post-induction or consolidation chemotherapy of AMLpatients who were registered with the Oncology Clinics of the Aga Khan University Hospital, Karachi. High WT1 burden (\u3e 5000 copies/ml) in 2 patients was indicative of early recurrence of the disease along with shorter disease-free and overall survival. Low WT1 expression (\u3c 200 copies/ml) in 2 patients after induction and consolidation therapy, respectively, was suggestive of better prognosis

Hepatitis B virus subgenotypes D1 and D3 are prevalent in Pakistan

<p>Abstract</p> <p>Background</p> <p>As the hepatitis B genotyping is important for assessing its clinical implications and geographical distribution, the sub-genotypes have been found useful for determination of specific genomic markers related to hepatocarcinogenesis. In Pakistan, there is no reported data on molecular evolutionary analysis of HBV. A study was, therefore, much needed to evaluate the spectra of mutations present in the strains prevalent here.</p> <p>Findings</p> <p>to confirm specificity of PCR typing, phylogenetic analysis of the pre-S1 region and the divergence was studied through 13 sequences of 362 bp (accession number <ext-link ext-link-type="gen" ext-link-id="EF432765">EF432765</ext-link> – <ext-link ext-link-type="gen" ext-link-id="EF432777">EF432777</ext-link>). A total of 315 serum samples, selected from HBsAg positive patients representing the major ethnic groups, residing in Karachi, Sindh were tested for genotyping. Genotype D (219/315) was found to be the most prevalent (70%) amongst our patients. The rest of the genotypes A and a mixture of A and D (AD) were distributed as 20%, and 10% respectively. Phylogenetic tree demonstrated clustering of 11 samples with subgenotype D1 sequences and the remaining two strains on a branch within D3 samples. All samples intermixed with strains from other countries and were found to be closely related to Indian, Iranian and Egyptian HBV strains with 98.7 – 99.0% homology.</p> <p>Conclusion</p> <p>This study confirms the predominance of genotype D in southeastern Asia and presence of subgenotypes DI and D3 in the Pakistani infected patients. More studies are required to investigate the reason for fewer inclusions of D3 compared to the D1 in Pakistani HBV strains.</p

Clinical presentation and genotype of hepatitis delta in Karachi

Aim: To assess the clinical presentation and genotypes of delta hepatitis in local population. Methods: In this prospective study, 39 consecutive patients who were positive for HBsAg and hepatitis D virus (HDV) antibody were included. The patients were divided in two groups on the basis of presence or absence of HDV RNA and a comparative study was done. Genotype of HDV was determined in PCR positive patients. Results:Overall there is male dominance, in which 34 patients out of 39 (87.2%) were male. Twenty (51%) patients were from the adjacent areas of three provinces; Sindh, Punjab and Balochistan indicating the higher prevalence of delta hepatitis in this mid region of Pakistan. Patients of all age groups were affected with delta hepatitis (median 31.5 years, range 12-75). HDV RNA was detectable in 23 patients (59%). All the HDV strains belonged to genotype I. HBV DNA was detectable only in 3 cases who were also HBeAg and HDV RNA positive. Patients with detectable HDV RNA were younger than patients with undetectable RNA; mean age 29.7 +/- 12.8 years vs 36.8 +/- 15.2. There were no statistically significant differences in the clinical presentation and routine biochemical profile of patients with detectable or undetectable HDV RNA. Clinical cirrhosis was present in 19 (49%) patients; 12 with detectable RNA and 7 with undetectable HDV RNA (P = 0.748). Decompensated disease was seen in eight patients; five and three respectively from each group. Four patients with undetectable RNA and two patients with detectable RNA had normal ALT and ultrasound abdomen. Conculsion: HDV may infect at any age, usually young adult males. Genotype I is prevalent. With time some of the patients become HDV RNA negative or asymptomatic carrier. Most of the patients have suppressed HBV DNA replication. Significant numbers of patients have cirrhosis