Mutation Spectrum and Genotype-Phenotype Analyses in a Pakistani Cohort With Hemophilia B.

This study aimed to (1) identify F9 genetic alterations in patients with hemophilia B (HB) of Pakistani origin and (2) determine the genotype-phenotype relationships in these patients. Diagnosed cases of HB were identified through registries at designated tertiary health-care centers across the country. Consenting patients were enrolled into the study. The factor IX (FIX) coagulation activity (FIX:C) and key clinical features were recorded. Direct sequencing of F9 was carried out in all patients. All the variants identified were analyzed for functional consequences employing in silico analysis tools. Accession numbers from National Center of Biotechnology Information ClinVar database were retrieved for the novel variants. Genotype-FIX:C relationships were determined followed by FIX:C clinical phenotype assessment. A total of 52 patients with HB from 36 unrelated families were identified, which mainly comprised patients with moderate HB (n = 35; 67.3%). Among these, 35 patients from 22 unrelated families could be contacted and enrolled into the study. Missense variants were the most frequent (58.8%), followed by nonsense variants (17.6%). A missense, a short insertion, and a nonsense novel variants in exon 2, 6, and 7, respectively, were also identified. The disease manifested FIX:C heterogeneity in relation to the corresponding mutation in a significant number of cases. Clinical phenotype heterogeneity was also observed in relation to FIX:C-based severity assessment. We concluded that the registered FIX-deficient population of Pakistan mainly comprises moderate HB. F9 mutation spectrum in Pakistani patients with HB is heterogeneous. The HB population of Pakistan manifests a significant amount of genotype-FIX:C and FIX:C-clinical phenotype heterogeneities.

Biallelic variants in LINGO1 are associated with autosomal recessive intellectual disability, microcephaly, speech and motor delay.

PURPOSE: To elucidate the novel molecular cause in two unrelated consanguineous families with autosomal recessive intellectual disability. METHODS: A combination of homozygosity mapping and exome sequencing was used to locate the plausible genetic defect in family F162, while only exome sequencing was followed in the family PKMR65. The protein 3D structure was visualized with the University of California-San Francisco Chimera software. RESULTS: All five patients from both families presented with severe intellectual disability, aggressive behavior, and speech and motor delay. Four of the five patients had microcephaly. We identified homozygous missense variants in LINGO1, p.(Arg290His) in family F162 and p.(Tyr288Cys) in family PKMR65. Both variants were predicted to be pathogenic, and segregated with the phenotype in the respective families. Molecular modeling of LINGO1 suggests that both variants interfere with the glycosylation of the protein. CONCLUSION: LINGO1 is a transmembrane receptor, predominantly found in the central nervous system. Published loss-of-function studies in mouse and zebrafish have established a crucial role of LINGO1 in normal neuronal development and central nervous system myelination by negatively regulating oligodendrocyte differentiation and neuronal survival. Taken together, our results indicate that biallelic LINGO1 missense variants cause autosomal recessive intellectual disability in humans.

Diagnosis and phenotypic assessment of Pakistani Haemophilia B carriers.

OBJECTIVES: 1: To assess the diagnostic utility of three polymorphisms (DdeI, XmnI and TaqI) and direct sequencing in haemophilia B (HB) carrier detection in Pakistani families. 2: To compare phenotypes of HB carriers with those of healthy females. METHODS: The study was conducted from March 2014 till February 2016 at Khyber Medical University Peshawar and National Institute of Blood Diseases, Karachi. Individuals from HB families of Khyber Pakhtunkhwa (KP) and Federally Administered Tribal Areas (FATA) with known F9 mutation in the proband were enrolled into the study. FIX activity (FIX: C) levels were determined in all the participants. Bleeding scores (BS) and complete blood counts were performed in the female participants. Linkage analysis followed by targeted Sanger sequencing was carried out in all the study participants. Heterozygosity rate was determined for each polymorphism. Healthy females and the carrier groups were compared for bleeding phenotypes. RESULTS: A total of 30 males and 48 females from 13 HB families were studied. The polymorphisms had a low heterozygosity rate. Direct sequencing determined the carrier status in all cases. The mean FIX: C was reduced whereas BS was raised in the carriers when compared with healthy females. A significant raise in white blood cells (WBCs) count was observed in the carriers. CONCLUSION: The three polymorphisms have a low heterozygosity rate in HB families from KP and FATA. Sanger sequencing is conclusive in determining carrier status in all the cases. FIX: C is low and BS is raised in the HB carriers in comparison to that of normal females. The mean WBCs count is significantly higher in the HB carriers than the normal females.

Sensitive detection of pre-existing BCR-ABL kinase domain mutations in CD34+ cells of newly diagnosed chronic-phase chronic myeloid leukemia patients is associated with imatinib resistance: implications in the post-imatinib era.

BACKGROUND: BCR-ABL kinase domain mutations are infrequently detected in newly diagnosed chronic-phase chronic myeloid leukemia (CML) patients. Recent studies indicate the presence of pre-existing BCR-ABL mutations in a higher percentage of CML patients when CD34+ stem/progenitor cells are investigated using sensitive techniques, and these mutations are associated with imatinib resistance and disease progression. However, such studies were limited to smaller number of patients. METHODS: We investigated BCR-ABL kinase domain mutations in CD34+ cells from 100 chronic-phase CML patients by multiplex allele-specific PCR and sequencing at diagnosis. Mutations were re-investigated upon manifestation of imatinib resistance using allele-specific PCR and direct sequencing of BCR-ABL kinase domain. RESULTS: Pre-existing BCR-ABL mutations were detected in 32/100 patients and included F311L, M351T, and T315I. After a median follow-up of 30 months (range 8-48), all patients with pre-existing BCR-ABL mutations exhibited imatinib resistance. Of the 68 patients without pre-existing BCR-ABL mutations, 24 developed imatinib resistance; allele-specific PCR and BCR-ABL kinase domain sequencing detected mutations in 22 of these patients. All 32 patients with pre-existing BCR-ABL mutations had the same mutations after manifestation of imatinib-resistance. In imatinib-resistant patients without pre-existing BCR-ABL mutations, we detected F311L, M351T, Y253F, and T315I mutations. All imatinib-resistant patients except T315I and Y253F mutations responded to imatinib dose escalation. CONCLUSION: Pre-existing BCR-ABL mutations can be detected in a substantial number of chronic-phase CML patients by sensitive allele-specific PCR technique using CD34+ cells. These mutations are associated with imatinib resistance if affecting drug binding directly or indirectly. After the recent approval of nilotinib, dasatinib, bosutinib and ponatinib for treatment of chronic myeloid leukemia along with imatinib, all of which vary in their effectiveness against mutated BCR-ABL forms, detection of pre-existing BCR-ABL mutations can help in selection of appropriate first-line drug therapy. Thus, mutation testing using CD34+ cells may facilitate improved, patient-tailored treatment.