1. Next-generation Sequencing MATERTILAS AND METHODS
Autosomal recessive polycystic kidney disease (arpkd) an overview with local perspective
Burhan M. Edrees1, 7, €, Mohammad Athar1, 2, €, Faisal A Al-Allaf1, 2, 3, €, Mohiuddin M. Taher1, 2, Wajahatullah Khan4, Abdellatif Bouazzaoui1, 2, Naffaa Al-Harbi5, Ramzia safar6, Howaida Al-Edressi6, Khawala Alansary7, Abulkareem Anazi7, Naji Altayeb7, 2, 3, Muawia A. Ahmed8, Zainularifeen Abduljaleel1, 2, €
Department of Medical Genetics, Faculty of Medicine, Umm Al-Qura University, P.O. Box 715, Makkah 21955, Saudi Arabia
Science and Technology Unit, Umm Al Qura University, P.O. Box 715, Makkah 21955, Saudi Arabia
Molecular Diagnostics Unit, Department of Laboratory and Blood Bank, King Abdullah Medical City, Makkah 21955, Saudi Arabia
Department of Basic Sciences, College of Science and Health Professions, King Saud Bin Abdulaziz University for Health Sciences, PO Box 3124, Riyadh 11426, Saudi Arabia
Department of Pediatric, King Faisal Specialist Hospital and Research Centre, P.O. Box 40047, Jeddah 21499, Saudi Arabia
Madinah Maternity and Children’s Hospital, P.O. Box 5073, Madinah 42318, Saudi Arabia. 
King Fahad Medical City, P.O. Box 59046, Riyadh 11525, Saudi Arabia
King Salman Armed Forces Hospital, PO box 100, Tabuk, Saudi Arabia 
€ Authors Contributed Equally to this Manuscript and shared 1st authorship
INTRODUCTION
RESULTS
Capillary sequencing
Autosomal recessive polycystic kidney disease (ARPKD) is a severe hereditary form of genetic disorder of the renal system which affects ~1:20,000 live-born children. The disease is responsible for a 30–40% mortality rate in the prenatal and neonatal children (Zerres et al., 1995). Affected individuals suffer hypertension, gastroesophegial bleeding, nephromegaly, oliguohydramnion, still birth, respiratory distress, fibrosis of the liver and renal failure (Gunay-Ayguna et al., 2011). The primary causative defect in ARPKD patients is mutations or deletions in the transmembrane protein encoding gene known as PKHD1 which is located at chromosome 6p12.2 (Bergmann et al., 2003). This gene spans 472 kb and including 67 exons that encodes 4074 amino acids protein, known as polyductin/fibrocystin (Ward et al., 2003). ARPKD is a recessive disorder with heterozygote ‘carriers’ being phenotypically normal.
The high incidence of consanguinity marriages in Saudi population which exceeding 54% suggesting that the incidence might be high (Mattoo et al., 1994; Mattoo et al 1998). Unfortunately, the spectrum of mutations causing ARPKD in Saudis, which is the prerequisite for prenatal and pre-implantation prevention regime, is not yet clearly understood (Gigarel et al., 2008). There are not many tools available for the neonatal or prenatal ARPKD diagnosis, and the only test is fetal sonography to check the enlargement of both kidneys as well as oligohydramnios (Zerres et al., 1988). Due to poor prognosis of early manifestations of ARPKD, there is a strong demand for the development of the genetic test for this disease and for carrier detection of ARPKD. Therefore, the aim of this proposal is to determine types and nature of mutations in the PKHD1 gene and its frequencies in clinically diagnosed Saudi ARPKD patients and to develop a molecular genetic testing for their detection. This genetic method will offer a fast, easy, and cost effective mutation screening method that can also be applied in the future for prenatal and pre-implantation genetic diagnosis to families at risk of having a child with ARPKD (Zerres et al., 2004).
Novel missense and known silent variants identified in ARPKD patients.
Known missense and silent variants identified in ARPKD patients.
Novel missense variant p.(Trp664Gly) identified at the PKHD1 Exon 21. A) DNA sequence from a healthy family control (WT) and heterozygous patient sample. B) Representative PCR gel image of PKHD1 exon 21 showing single specific band of correct size.
Known missense variant p.(Val3193Ile) identified at the PKHD1 Exon A) DNA sequence from a healthy family control (WT) and heterozygous patients samples. B) Representative PCR gel image of PKHD1 exon 58-3 showing single specific band of correct size.
Next-generation Sequencing
Capillary validation. The NGS resulted variants confirmed by capillary sequencing method. Pink colors represent a reference of mRNA transcript and variants row were highlighted. A) A variant c.4870C>T, p.(Arg1624Trp) was in patients 2, 6, 9, 10, 11, 14, 15, 16. B) A novel variant c.10628T>G, p.(Leu3543Trp) in-patient eight. C) A variant c.2027C>G, p.(Pro676Arg) in-patient one. D) A variant c.5725C>T, p.(Arg1909Trp) in a patient one. E) A variant c.1736C>T, p.(Thr579Met) in patient four.
Overview of targeted pkhd1 variants (Novel and known) in different samples by Ion-Torrent PGM
OBJECTIVE
Identification of families, from several Saudi regions with suspected Autosomal recessive polycystic kidney disease (ARPKD).
To identify the genetic mutations causing ARPKD among Saudi Arabian children.
MATERTILAS AND METHODS
ARPKD samples collections :
Sample collection and genetic studies were performed in accordance with the Research Ethics Committee’s regulation.
A signed informed consent for genetic screening was obtained from their parents.
63 unrelated ARPKD patients were recruited for genetic screening of ARPKD at four referral hospitals from central (Riyadh), western (Makkah, Medina, Jeddah) and northern (Tabuk) regions of Saudi Arabia.
2-5 mls of whole peripheral blood were collected from patients
Parameters for Sanger Sequencing:
DNA extraction : MagNA Pure automated instrument (Roche)
PCR (PKHD1 gene) : Hot Start PCR Kit (Qiagen)
Size and Quality of a band : Agarose gel electrophoresis
PCR product purification : Agencourt AMPure XP magnetic beads
Cycle sequencing : Big Dye terminator v3.1 cycle sequencing kits
5. Sequenced product purification : Big Dye X-terminator purification kit
6. Sequencing : 3500 Genetic Analyzer (ABI)
Data Analysis : Sequencing Software v5.4 (ABI)
Next Generation Sequencing (NGS) by Ion Torrent PGM:
We have customized TargetSeq kit for PKHD1, PKD1 and PKD2 genes by Applied Biosystem.
Identified variants in pkhd1 gene. A) In patient eight have one novel variant c.10628T>G, p.(Leu3543Trp). B and C) In patient one have two known variants c.5725C>T, p.(Arg1909Trp) and c.2027C>T, p.(Pro767Arg). D) In patient four have one known variant c.1736C>T, p.(Thr579Met). E) In patients P2, P6, P9, P10, P11, P14, P15, and P16 have one common variant in the Saudi Patients, which are reported but in other populations.
CONCLUSION
So far, we have collected 63 ARPKD patients samples from unrelated families
We have established and optimized DNA sequencing testing
We identified two novel mutations in PKHD1 gene causing ARPKD in Saudi population
We have also identified five known deleterious mutations in PKHD1 gene causing ARPKD in Saudi population
NGS experimental workflow
ACKNOWLEDGEMENT
We are indebted to probands and family members for their cooperation , and thankful for the academic department of the security forces hospital in MAKKAH (designing the poster). The work is supported by a Grant Number 10-BIO , the Long-Term National Plan for Science, Technology and Innovation, KACST.