Clinical characterizations and genetic analysis of metabolic liver disorders in local Population

Abstract

Most of metabolic liver diseases are caused by mutation in a gene/s encoding an enzyme or transport protein and biochemical analysis of serum analytes do not provide specific diagnosis. Specific and differential diagnosis therefore require liver biopsy for enzyme assay or histological analysis for protein transporters, which is performed in few laboratories around the world and require shipment of samples under dry ice which adds to the cost and is not often feasible for long distances. Thus, such diseases remain undiagnosed and hence untreated mainly in low-income countries with poor health facilities like Pakistan. To study the clinical characterization and molecular analysis of metabolic liver disorders in Pakistan, ethical approval was approved from Bioethical review board of Quaid-i-Azam University, Islamabad Pakistan. In the present study cases of three metabolic liver disorders i.e., Wilson’s disease (WD), Glycogen storage disease type 1a (GSD 1a) and Intrahepatic cholestasis of pregnancy (ICP) were enrolled after informed consent. Inherited mutations in the ATP7B gene cause Wilson's disease (WD), which is an autosomal recessive disorder. Diagnosis can be challenging because of wide range of the ages affected and the clinical heterogeneity that includes neuropsychiatric and hepatic signs. Patients suffering from WD can face severe form of disabilities or even decease. So, the inclusive purpose of this research was characterization of WD cases on clinical and genetic basis from Pakistani population. 80 patients with WD were included in study from the Pediatric Department and Liver Unit of Children Hospital Lahore (CHL), Pakistan institute of medical sciences (PIMS), over the period January 2018 to January 2020. Clinical data was recorded for demographic and statistical analysis of clinical variants using SPSS 20.0. Patients showed a wide range of clinical variability, with some common diagnostic features such as lower serum levels of ceruloplasmin and elevated 24-hour urinary copper defecation. Mean age at the diagnosis was 11.3 years. Parental cousin marriage was observed among 80 patients. Kayser-Fleischer ring was observed in 47 patients in their eyes due to copper deposition in decement membrane of the cornea. Family history of WD was observed in 47.5%. Statistical analysis showed significance between ix stage of illness and deceased-alive status of patient. of the cases. Association of stage of disease with risk factors like age, therapy received, and symptom reversal showed significant relationship. Based on availability of funds and willingness to participate in molecular analysis, blood samples of 20 patients were collected and investigated for PCR based Sanger sequencing of both the forward and reverse directions of the exon 1, 2, 3, 4, 5, 8 & 13 of the ATP7B gene for disease causing variants, the software PROVEAN, MutationTaster, Polyphen 2, SIFT, and HSF were used to predict the pathogenic effects of the variants that were found. Study predicted the pathogenic effects of the identified mutations. The study identified 22 variants in selected exons and intron-exon boundaries, 7 disease causing variants and 15 polymorphisms. Out of 7 identified disease-causing variant (6 homozygous and 1 heterozygous), 3 (p.V761E, p.I976N, p.I774L are novel and 4 (L227Yfs*35, p.D765N, p.A1003A and p.S986T) are reported. Out of 15 polymorphisms 5 are known and 10 are novel. The results of the investigation revealed the clinical presentation of WD patients in Pakistan and identified sequence variants in the ATP7B region that were tested. Type I glycogen storage disease (GSD I) is a rare autosomal recessive disease; hence it is the most likely diagnosis when a baby is born with a genetic problem. Glucose-6 phosphatase complex malfunction causes glycogen storage disorder type Ia (GSD Ia), an extremely rare autosomal recessive congenital mistake in carbohydrate metabolism (G6PC). Glycogen storage disorder type Ia (GSD Ia; Von Gierke Disease; MIM #232200) is caused by deficiency in this enzyme and is typically characterized by the hypoglycemia, hepatomegaly, hyperuricemia, lactic acidemia, and hyperlipidemia. Disease causing variants in the G6PC gene, located on the chromosome 17q21 result in glycogen storage disease type Ia (GSD Ia). Age of onset of GSD 1a ranges from 0.5 to 25 years with presenting features including hemorrhage, hepatic, physical and blood related abnormalities. The overall aim of present study was clinical and genetic characterization of GSD 1a cases from Pakistani population. This study included forty GSD1a cases presenting with heterogeneous clinical profile including hypoglycemia, hepatomegaly, lactic acidosis i.e., pH less than 7.2, hyperuricemia, seizures, epistaxis, hypertriglyceridemia (more than180 mg/dl) and sometimes short stature. All coding exons and intron-exon boundaries of G6PC gene were screened to identify pathogenic variant in x 20 patients based on availability of funds and willingness to participate in molecular analysis, on availability of DNA samples. The PCR-Sanger sequencing technique was used for mutation analysis, and the softwares PROVEAN, MutationTaster, Polyphen 2, SIFT, and HSF were used to predict the pathogenic effects of the variants that were found. Overall, 21 variants were detected including 8 novel disease causing variants i.e., p. Ala37Pro, p. Asp69Asn, p.Gln82Lys, p.Thr108Pro, g.50_51insT, p.Gln24Pro, p.Val45Leu and p.Cys284Tyr in the screened regions of G6PC gene. Out of 13 identified polymorphisms, 4 were identified in heterozygous condition while 9 were found in homozygous condition. This study revealed clinical presentations of GSD1a cases from Pakistan and identification of novel disease-causing sequence variants in coding and intron-exon region boundaries of G6PC gene. Intrahepatic cholestasis (ICP), medically known as, obstetric cholestasis is pregnancy specific liver disease in which onset of symptoms occurs in third term. General pruritus, irregular liver function test and elevated serum bile acid are presenting symptoms. Increased load of pregnancy specific hormones and their metabolites obstructs the function of canalicular transporter proteins. Consequently, bile acid accumulates in blood serum. Intrahepatic cholestasis, although is of less significance for maternal health as it resolves after delivery, but it poses serious fetal risks. ICP has 0.2-2% prevalence worldwide. In Pakistan, 3% prevalence has been reported. ICP also exhibits familial clustering in certain cases that indicates role of the genetic factors. Mutations in canalicular biliary transport proteins disturb the mechanism of bile synthesis and flow. The candidate genes investigated for pathogenicity of ICP are: farnesoid X receptor ATP8B1 (FIC1), bile salt export pump (BSEP), ABCB11 and phospholipid translocator ABCB4 (MDR3). To understand the molecular basis of ICP in our population, we screened known variants of exon 6 and exon 14 of ABCB4 gene in 50 ICP patients presented at Wah General Hospital, during a course of six months (June 2019-November 2019). Preterm birth, meconium staining of amniotic fluid and stillbirth are some of the perinatal outcomes that have been documented alongside maternal pruritus and elevated blood bile acids. A molecular analysis of genetic variants of ICP was done by ARMS (Amplification Refractory Mutation System) PCR in 50 cases and 50 controls for the identification of known variants in exon 6 and 14 of ABCB4 gene. The study identified disease causing variants i.e., c.504 C>T; in xi 17 cases, to be significantly associated with the disease. The variant c.1686 A>G showed non-significant association. This study gives a way for improved understanding of molecular mechanism elucidating the phenotypic variability and genetic heterogeneity among patients with metabolic disorders i.e., Wilson’s disease, Glycogen storage disease type 1a and Intrahepatic cholestasis of pregnancy. There are gaps in current knowledge about disease risk association of single nucleotide polymorphisms with physiological factors, potential target molecular pathway/s of metabolic dysfunction predisposing to specific clinical endpoints of liver dysfunction and their association to genetic attributes. The outcomes of this study provide further knowledge of genetic variants from our Pakistani population. Genetic counselling was provided to all families affected with WD and GSD 1a to refrain from cousin marriages that would help in decreased incidence of these devastating conditions in upcoming generations as ICP can affect subsequent pregnancies of affected women, therefore all the affected females enrolled in this study were provided with counseling to monitor the symptoms and seek proper screening in subsequent pregnancies. Furthermore, our findings help the clinicians to understand involvement of genetic factors and clinical heterogeneity of these conditions in our population, so that they may recommend the suspected cases for genetic analysis to get differential diagnosis for proper patient management. However further genetic studies are required to understand genetic basis of metabolic liver disorders, identification of prevalent sequence variants for establishment of cost effective molecular genetic tests for our local population