Clinical and Genetic Description of Hereditary Chronic Pancreatitis in Pakistani Children

Background/Aims: The purpose of this study was to identify the spectrum and frequency of pathogenic variants as well as the clinical and genetic insight of hereditary chronic pancreatitis in Pakistani children. Materials and Methods: The deoxyribonucleic acid of affected probands of 44 unrelated Pakistani families, having hereditary chronic pancreatitis-affected children, were subjected to massive parallel sequencing for candidate reported genes (SPINK1, PRSS1, CFTR, CPA1, CTRC, CBS, AGL, PHKB, and LPL). Data were analyzed using different bioinformatics tools for the variants and in-silico analysis. All the identified variants were validated by direct sequencing of the targeted exons in the probands and their parents. Results: There were 50 patients included in this study with confirmed hereditary chronic pancreatitis. Nine known mutations in SPINK1, PRSS1, CFTR, CTRC, CBS, and AGL genes, and 10 novel variants in LPL, CFTR, CTR, and PHKB genes were identified. The identified variants were found in heterozygous, compound heterozygous, and trans-heterozygous forms, with rare allele frequency in the normal population. The novel variants were [c.378C>T(p.Lys126Asn) and c.719G>A(p.Arg240Gln) in CTRC, c.586-3C>A and c.763A>G(p.Arg255Gly) in CPA1, c.1160_1161insT(p.Lys387Asnfs*26), c.784C>T(p.Gln262*), c.1139+1G>A, c.175G>A(p.Gly59Arg) in LPL, c.388C>G(p.leu130val) in CFTR, and c.2327G>A(p.Arg776His in PHKB)]. The phenotypic characteristics were variable and correlated with the relevant variant. Conclusions: The genetic composition plays a significant role in the predisposition of hereditary chronic pancreatitis. The clinical presentation varies with the genetic determinant involved. This information would help in building up a diagnostic algorithm for our population that can be used for genetic screening services in affected cohorts.


INTRODUCTION
Hereditary pancreatitis is one of the frequently described causes of recurrent acute and chronic pancreatitis in children in addition to hepatobiliary diseases.The incidence of hereditary pancreatitis among chronic pancreatitis cases is 0.3 to 0.5/100 000. 1,2There is no gender bias, but racial preponderance does exist, and it is more frequently reported in black ethnic origin in the West. 2 The clinical presentation of hereditary chronic pancreatitis (HCP) is not different from the chronic pancreatitis of other etiologies.Abdominal pain with or without vomiting is the major clinical presentation followed by failure to thrive, steatorrhea, and family inheritance. 3,4omplications are part of chronic pancreatitis and invariably happened in the affected children, but with appropriate management of their various clinical manifestations, most of them can have a good quality of life. 5st of the cases of recurrent acute pancreatitis and chronic pancreatitis in the past were considered idiopathic, but with the recent advances in genetic and autoimmune studies, the scenario has changed.Biochemical parameters, including serum amylase and lipase, are not helpful in these children owing to the chronic nature of the disease and severe atrophy of pancreatic tissue.Radiological modalities are most valuable in delineating the structural changes in HCP. 6ypsin is activated intestinally by serine protease enteropeptidase while its auto-activation (trypsin-mediated trypsinogen) occurs in the pancreas.Mutations in cationic trypsinogen that increase auto-activation are strong risk factors for chronic pancreatitis, typically associated with hereditary pancreatitis.Protective mechanisms that cut down trypsinogen activation in the pancreas are either trypsin inhibition or trypsinogen degradation.Thus, pancreatitis is caused by uncontrolled trypsin activity either by increasing the trypsinogen activation to trypsin or by impairing the protective trypsinogen degradation and/or trypsin inhibition. 7e genetic analysis for the diagnosis of HCP is a sensitive and powerful tool.Different genetic mutations have been described in the literature with variable presentation and the majority of them have an autosomal dominant pattern of inheritance.The common genes involved in HCP are PRSS1 (prevent proper auto inactivation of enzyme), SPINK1, CTRC (regulate trypsin inhibition and inactivation), CFTR (clear trypsin from the Pancreas), CPA1, and several other trypsin-dependent genetic causes. 1,2,8The clinical heterogeneity of pancreatic disease is at least partly due to allelic heterogeneity.Irrespective of the advancement in molecular biology, the underlying genetic cause of almost 30% of the affected families having hereditary pancreatitis is still unknown.The pathogenic variants of hereditary pancreatitis have not been evaluated in Pakistani children, so we aimed to identify the spectrum and frequency of pathogenic variants as well as the clinical and genetic insight into HCP in Pakistani children.

MATERIALS AND METHODS Patients Recruitment
This is a cross-sectional, descriptive retrospective study of children having chronic pancreatitis with the demonstration of genetic variants.The study was conducted at the Department of Pediatric Gastroenterology, Hepatology and Nutrition from January 2017 to December 2020.During this period, 78 Pakistani children clinically diagnosed with chronic pancreatitis were enrolled for genetic analysis.This study was approved by the institutional review board of Children's Hospital and University of Child Health Sciences, Lahore Pakistan (Approval No: 2019-22-CHICH) and conducted according to the principles of the Helsinki Declaration.Informed written consent from parents /guardians was obtained prior to their enrollment in the study.
The inclusion criteria were adopted from INSPPIRE definition of chronic pancreatitis. 8

INSPPIRE Definition of Chronic Pancreatitis
It requires at least 1 of the following: 1. Abdominal pain consistent with pancreatic origin with imaging findings suggestive of chronic pancreatitis.

Evidence of exocrine pancreatic insufficiency with
imaging findings suggestive of chronic pancreatitis.3. Evidence of endocrine insufficiency with imaging findings suggestive of chronic pancreatitis.4. Surgical or pancreatic biopsy specimens demonstrating histopathologic features compatible with chronic pancreatic (CP).

Radiological Modalities
Imaging examination including ultrasound abdomen, computed tomography (CT), magnetic resonance imaging, and magnetic resonance chola ngiop ancre atogr aphy (MRCP) was performed to identify the findings suggestive of chronic pancreatitis and included pancreatic parenchymal changes like heterogeneity, pancreatic calcifications, dilatation or irregularity of main pancreatic duct, or pancreatic atrophy.

Genetic Testing
Genomic deoxyribonucleic acid (DNA) was extracted from ethylenediamine tetraacetic acid (EDTA)-treated peripheral blood samples by using a QIAamp DNA Blood Mini kit (Qiagen, Hilden, Germany).Next-generation sequencing (NGS) was performed at CENTOGENE (Germany).Briefly, the extracted DNA was enzymatically fragmented and regions of interest were selectively enriched using capture probes and targeted against coding regions of ~6700 genes with known clinical significance.Libraries were generated with Illumina-compatible adaptors and sequenced on an Illumina platform.The evaluation was focused on coding exons along with flanking ±10 intronic bases within the captured region.Variants identified by NGS were Sanger sequenced to exclude any artifacts.Two independent copy number variation (CNV) callers were used to determine CNVs within the panel genes from the NGS data.All clinically relevant CNVs were confirmed with an   1 and Table 2.  2).

In-Silico Protein Analysis of Novel Mutations
Protein simulation and annotations were performed using 3 PyMol was used to physically show the interactions and difference of amino acids in wild and mutant form, as shown in Figure 1, and signifies the changes in interaction with the neighboring amino acids.The △△G energy was calculated to determine whether the protein structure is rendered unstable due to the mutation.Overall stability is calculated from atom potentials and torsion angle potentials.In case of unfavorable torsion angles, the atom potentials may have a higher impact on stability, which results in a stabilizing mutation (Tables 3 and 4).
We also measured the torsion angle changes, the solvent accessibility (%), and depth (Å).The c.378G>T(p.Lys126Asn) variation which did not reduce the stability of protein and this patient (PT 14) also has other mutations which are dominant and explain the single mutation affect.
We found 2 types of heterozygous pathogenic PRSS1 variants in 12 pediatric patients, including c.365G>A(p.Arg122His) (n = 10), and copy number variable (n = 2).Among patients with CNV (n = 1), CFTR c.388C>G(p.Leu130Val) (n = 1) was also detected.We   They exhibit high penetrance and follow the autosomal dominant pattern of pancreatitis.There was no genotype-phenotype correlation or its effect on the age of symptom onset, among our cohort.

DISCUSSION
The HCP is one of the genetically determined conditions with different modes of inheritance seen in the pediatric age group.It is also a common cause of chronic pancreatitis in children, followed by hepatobiliary and idiopathic causes. 9It results from an imbalance of proteases and their inhibitors in the pancreatic parenchyma thus forming a complex syndrome of varying signs and symptoms including multiple causative pathways that converge into similar phenotypic features. 10The incidence of hereditary pancreatitis is 0.3 to 0.5/100 000 cases in the West; however, due to a lack of epidemiological studies, the exact prevalence in the Pakistani population is not known.In this hospital-based study, 64.1% of children had been genetically identified positive among the clinically diagnosed chronic pancreatitis cohort.The gender predilection is the same in pediatric patients though males are more affected in adult literature because of alcohol consumption. 11In our study, females are affected more than males.Racial difference does exist in this morbid condition, and Black ethnic origin outnumbers the white population in the West. 12reditary chronic pancreatitis affects children at an early age leading to different manifestations later. 13,14n our study, there is a delay in diagnosis of about 3.5 years before the confirmation of a diagnosis of HCP could be made.The clinical spectrum of presentation of HCP is no different from chronic pancreatitis of other etiologies. 2,13,15Abdominal pain, vomiting, failure to thrive, exocrine insufficiency, and endocrine issues are the main presenting features in the international literature and are similar to our study, as shown in Table 1. 4,16The radiological finding of chronic pancreatitis is the primary lead in addition to the clinical background to further investigations in these children.Common radiological findings are calcification in pancreatic parenchyma and dilated and beaded pancreatic duct on ultrasound, CT, or MRCP. 5 There are a number of mutations identified in recent years in HCP.The majority of them are inherited as an autosomal dominant pattern.Two patients had duplication in PRSS1 gene; in 1 patient, there is a gain of 1 copy, encompassing exons 1-5 while in the other, there is a gain of 2 copies, encompassing the whole PRSS1 gene.Gain-of-function pathogenic PRSS1 variants, including pathogenic missense mutations and pathogenic CNVs such as gene duplication or triplication, have already been reported as the most common PRSS1 disorder. 19Schnúr et al 20 reported that c.410C>T (p.Thr137Met), c.508A>G (p.Lys170Glu), and c.623G>C (p.Gly208Ala) variants in PRSS1are rare and predominant in subjects of Asian origin.None of our patients had these variants rather they had the most commonly "classic pathogenic variant" c.365G>A (p.Arg122His).
Four children with HCP, had unusual mutation identified as lipoprotein lipase deficiency (LPL), c.1160_1161insT, and c.175G>A(p.Gly59Arg) variants had autosomal recessive pattern while c.784C>T(p.Gln262*), and c.1139+1G>A had autosomal dominant pattern which is similar to international literature. 21c.175G>A(p.Gly59Arg) variant was found in combination of c.2327G>A(p.Arg776His) variant in PHKB1.They were being managed as hyper trigl yceri demia -indu ced chronic pancreatitis.Among these 4 children, 2 were sisters, and the other 2 belonged to different families.
Chronic pancreatitis caused by CFTR is inherited in an autosomal recessive or complex manner due to compound heterozygosity for CFTR or a combination of defects in CFTR and other genes. 22Cationic trypsinogen mutation (PRSS1) generally does not require added risk factors, but others like SPINK1 and CFTR are considered disease modifiers and require additional factors for manifestation. 23The risk factors identified in these children are abdominal trauma, hypertriglyceridemia, and familial pattern.In addition to these risk factors, the transheterozygous mutation is also considered a risk factor like CFTR and SPINK1 co-mutation. 18,23Diagnosing early chronic pancreatitis remains challenging in pediatrics as in adults.Previous studies reported a 30%-73% association of genetic mutations with pancreatitis. 24The mode of presentation may be unusual in children owing to the presence of unique genetic mutations.
Hereditary chronic pancreatitis accounts for approximately 20%-25% of all cases in childhood.About 4 probands had no family history of chronic pancreatitis, although the mutation was inherited in all cases by at least 1 parent, indicating a low penetrance of these mutations.This finding is in contrast to the high penetrance reported for the c.365G>A(p.Arg122His) mutations in PRSS1.Thus, trypsinogen mutations display considerable variability with respect to penetrance.Although c.365G>A(p.Arg122His) mutation has also been in "sporadic" cases of chronic pancreatitis without a family history.The high penetrance reported may reflect more the penetrance in certain chronic pancreatitis families (sharing a similar genetic background) than the penetrance of these mutations in general. 25dical management is the cornerstone of treatment in this highly morbid condition.Most patients are managed with dietary restrictions, analgesics, pancreatic enzyme replacement therapy, and proton pump inhibitors in addition to insulin for endocrine complications and are similar to the current study.7][28] The majority of our children had a good quality of life on medical measures, but 20% of children required surgical interventions in the form of cyst gastrostomy and Puestow procedure for relief from pseudocyst and pain, respectively.
The novel mutations mapped in LPL, CFTR, CTRC, and PHKB genes show a potential for new finding in pancreatitis and their clinical-genetic importance for diagnostic and pharmaceutics.Although this study had a small sample size, it includes an expanded panel of genes in case of hereditary pancreatitis.This is a very important finding in the Pakistani cohort for further clinical-genetic criteria revision at the local level.
Limitation of our study includes the retrospective nature, single-center database, and potential missing of data could not be ruled out; however, an attempt is made to share the perspective of hereditary chronic pancreatitis in Pakistani children.We still need further cellular and drug testing studies to evaluate these new novel mutations.

CONCLUSION
This is the first-ever long clinical and genetic follow-up study on chronic pancreatitis disease in Pakistani children.Heterogeneous genetic factors play a significant role in disease manifestation.Genetic variants confer susceptibility to HCP, and which polymorphisms modify the prognosis and treatment response that leads to chronic pancreatitis will also be important in determining the timing of genetic testing and subsequent intervention.Micromanagement with painkillers, dietary modifications and exocrine/endocrine support keeps a good quality of life, but surgical intervention is definitely needed if medical measures fail.It shows genetic and clinical diagnosis, and follow-up is critical for accurate and personalized medication.Genetic testing in the future will therefore require the inclusion of many polymorphisms, and the results will require careful interpretation.It is conceivable that therapy will be targeted at preventing HCP through genetic predisposition.Supplementary Table 1.

Figure 1 .
Figure 1.Crystal structure and distance upon mutation are illustrated.

Table 1 .
Demographic, Risk Factors, Clinical Presentation, and Radiological Modalities in 50 Pakistani Patients with Hereditary Pancreatitis CFTR, and c.2327G>A(p.Arg776His in PHKB)] were found with a low rare allele frequency in normal population (Table

Table 2 .
Genetic Data of All Mutations of Known and Candidate Genes Found in our Cohort

Table 2 .
Genetic Data of All Mutations of Known and Candidate Genes Found in our Cohort (Continued) B, benign; D, damaging protein; DC, disease causing; gnomAD, genome aggregation database; Het, heterozygous; Hom, homozygous; P, polymorphism.

Table 2 .
Genetic Data of All Mutations of Known and Candidate Genes Found in our Cohort (Continued)

Table 3 .
Novel Mutation △△G and Torsion Angle Measurement

Table 1 .
The Demographic, Clinical Characteristics with Brief Management Summary