Unrelated Iranian Families Research Articles

Identification of novel and known genetic variants associated with hereditary hearing loss in iranian families using whole exome sequencing.

Hearing loss (HL) is a common sensory impairment worldwide, with genetic and environmental factors contributing to its occurrence. Next Generation Sequencing (NGS) plays a crucial role in identifying the genetic factors involved in this heterogeneous disorder. In this study, a total of 9 unrelated Iranian families, each having at least one affected individual who tested negative for mutations in GJB2, underwent screening using whole exome sequencing (WES). The pathogenicity and novelty of the identified variant was checked using various databases. Co-segregation study was also performed to confirm the presence of the candidate variants in parents. Plus, The pathogenicity of the detected variant was assessed through in silico analysis using a number of mutation prediction software tools. Among the 9 investigated families, hearing loss-causing genes were identified in 6 families. the mutations were observed in USH2A, CLRN1, BSND, SLC26A4, and MITF, with two of the identified mutations being novel. Discovering additional variants and broadening the range of mutations associated with hearing impairment has the potential to enhance the diagnostic effectiveness of molecular testing in patient screening, and can also lead to improved counseling aimed at reducing the risk of affected offspring for high-risk couples.

JAM2 variants can be more common in primary familial brain calcification (PFBC) cases than those appear; may be due to a founder mutation.

Mutations in JAM2 have been linked to ~ 2% of primary familial brain calcification (PFBC) cases. PFBC is a rare neurological disorder characterized by excessive calcium deposition in the brain. It causes movement disorders and psychiatric problems. Six other genes were identified as causing PFBC. However, the genetic basis of ~ 50% of PFBC cases remains unknown. This study presented the results of a comprehensive analysis of five unrelated Iranian PFBC families. Clinical and paraclinical features of all patients were recorded. Whole-exome sequencing (WES) was done on the DNAs of probands. Data was analyzed, and haplotypes were determined. WES identified two homozygous variants in JAM2 across four families: a novel variant, c.426dup:p.Ser143Leufs*23, in one family and a known mutation, c.685C > T:p.Arg229*, in the remaining three families. Haplotype analysis using six intragenic single-nucleotide polymorphisms (SNPs) in JAM2 revealed an identical haplotype in probands who carried the same mutation, whereas two other probands presented diverse haplotypes. Based on our results, p.Arg229* may be a founder mutation in the Iranian population. The variant has been detected in two out of seven other reported JAM2-related families who may originate from the Middle East and exhibit an identical haplotype. Even though this particular mutation may not be classified as a founder mutation, it does appear to be a hotspot, given that it has been observed in 45% of the 11 JAM2-associated families. Our study expanded the clinical features and mutation spectrum of JAM2 and revealed that mutations in JAM2 may be more common than previously reported.

The first reports of FA2H-associated neurodegeneration from two unrelated Iranian families.

NBIA (neurodegeneration with brain iron accumulation) is a diverse collection of neurodegenerative illnesses defined by iron accumulation in the basal ganglia. The fatty acid hydroxylase-associated neurodegeneration, or FAHN, is one of the uncommon subtypes of NBIAs, associated with inherited autosomal recessive mutations in gene coding the membrane-bound fatty acid 2 hydroxylase (FA2H) enzyme. Here, we report two cases with FAHN from two unrelated families from Iran confirmed by whole exome sequencing. FAHN is an uncommon variant of NBIA that may manifest as spastic paraparesis without signs of iron buildup on brain imaging. As a result, it should be taken into account while making a differential diagnosis of the hereditary spastic paraplegia (HSP) syndrome, especially in individuals who lack iron deposits.

Clinical and genetic characterization of neuronal ceroid lipofuscinoses (NCLs) in 29 Iranian patients: identification of 11 novel mutations.

Neuronal ceroid lipofuscinoses (NCLs) are neurodegenerative lysosomal storage diseases which are considered among the most frequent causes of dementia in childhood worldwide This study aimed to identify the gene variants, molecular etiologies, and clinical features in 23 unrelated Iranian families with NCL. In total, 29 patients with neuronal ceroid lipofuscinoses (NCLs), diagnosed based on clinical manifestations, MRI neuroimaging, and electroencephalography (EEG), were recruited for this study. Through whole-exome sequencing (WES), functional prediction, Sanger sequencing, and segregation analysis, we found that 12 patients (41.3%) with mutations in the CLN6 gene, 7 patients (24%) with the TPP1 (CLN2) gene variants, and 4 patients (13.7%) with mutations in the MFSD8 (CLN7) gene. Also, mutations in each of the CLN3 and CLN5 genes were detected in 2 cases and mutations of each PPT1 (CLN1) and CLN8 gene were observed in only 1 separate patient. We identified 18 different mutations, 11 (61%) of which are novel, never have been reported before, and the others have been previously described. The gene variants identified in this study expand the number of published clinical cases and the variant frequency spectrum of the neuronal ceroid lipofuscinoses (NCLs) genes; moreover, the identification of these variants supplies foundational clues for future NCL diagnosis and therapy.

NRXN3 mutations cause developmental delay, movement disorder, and behavioral problems: CRISPR edited cells based WES results

NRXN3geneencodesneurexin-III which is a Neural Cell Adhesion Molecule (NCAM) with important synaptic functions in the brain. Neurexin-III deficiency could affect synapse development, synaptic signaling and neurotransmitter release. Hitherto, there is no related disorder in the OMIM due to NRXN3 mutation. In this study, two unrelated Iranian families with homozygous (NM_001330195.2:c.3995G>A, p.Arg1332His) and compound heterozygous (NM_001330195.2:c.4442G>A, p.Arg1481Gln; c.3142+3A>G) variants in theNRXN3gene were detected for the first time. The proband of the first family manifested learning disability, developmental delay, inability to walk, and behavioral problems such as difficulty in social communication. Also, global development delay, intellectual disability, abnormal gait, severe speech problems, muscle weakness, and behavioral problems were observed in the affected individual in the second family. In addition, deciphering the pathogenicity of NRXN3 variants was done by functional studies such as CRISPR edited cells, in-silico analysis, and NGS results. All of these data together with phenotype similarity between observed phenotypes in our patients and manifested symptoms in the homozygousNrxn3α/β knockout mice, demonstrate the homozygous and compound heterozygous mutations of NRXN3 could cause a novel syndromic mendelian genetic disorder with autosomal recessive inheritance. The main phenotype of patients with neurexin-III deficiency includes developmental delay, learning disability, movement disorder, and behavioral problems.

A Study on the CLCN5 Gene in Iranian Patients: A Report of Novel and Recurrent Mutations

Introduction: Dent’s disease is an X-linked inherited renal tubular disorder characterized by proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis, rickets, and end-stage renal disease. Almost 60% of patients have causative mutations in the CLCN5 gene (Dent 1), and 15% of affected individuals have mutations in the OCRL1 gene (Dent 2). The aims of this study are to identify CLCN5 mutations in Iranian families with Dent’s disease and to characterize the associated clinical syndromes. Methods: We studied 14 patients from 13 unrelated Iranian families with a clinical diagnosis of Dent’s disease. Proteinuria was detected in all patients. Nephrolithiasis was found in 5 patient, and hematuria in 2 patients. Most of the affected individuals had nephrocalcinosis. PCR-sequencing for the CLCN5 gene was performed in all 14 patients. Next-generation sequencing (NGS) has also been performed in one patient who we did not find causative mutation. Results: We identified four different CLCN5 mutations including one missense mutation (c.731C>T), one nonsense mutation (c.100C>T), and two novel mutations, consisting of one frameshift mutation (c.1241_1242dupAA) and one splicing mutation (c.805-2A>G). We also identified one OCRL1 mutation, one splicing mutation (c.1466 + 1G>A), using NGS. Conclusion: This is the first report to characterize mutations in the CLCN5 gene in Iranian patients with Dent’s disease and expands the spectrum of CLCN5 mutations by reporting two novel mutations, c.1241_1242dupAA and c.805-2A>G.

Reporting Two Novel Mutations in Two Iranian Families with Cystic Fibrosis, Molecular and Bioinformatic Analysis

Cystic fibrosis is the most common heredity disease among the Caucasian population. More than 350 known pathogenic variations in the CFTR gene (NM_000492.4) cause CF. Herein, we report the outcome of our investigation in two unrelated Iranian families with CF patients. We conducted phenotypic examination, segregation, linkage analysis, and CFTR gene sequencing to define causative mutations. We found two novel mutations in the present study. The first one was a deletion causing frameshift, c.299delT p.(Leu100Profs*7), and the second one was a missense mutation, c.1857G>T, at nucleotide binding domain 1 of the CFTR protein. Haplotype segregation data supported our new mutation findings. Findings of this study expand the spectrum of CFTR pathogenic variations and can improve prenatal diagnosis and genetic counseling for CF.

Reporting Two Novel Mutations in Two Iranian Families with Cystic Fibrosis, Molecular and Bioinformatic Analysis

Cystic fibrosis is the most common heredity disease among the Caucasian population. More than 350 known pathogenic variations in the CFTR gene (NM_000492.4) cause cystic fibrosis. Herein, we report the outcome of our investigation of two unrelated Iranian families with cystic fibrosis patients. We conducted phenotypic examination, segregation, and linkage analysis, and CFTR gene sequencing to define causative mutations. Two novel mutations were found. One was a deletion causing frameshift, c.299delT p.(Leu100Profs*7) and the second one was a missense mutation, c.1857G>T at nucleotide binding domain 1 of the CFTR protein. Haplotype segregation data supported our new mutation findings. These findings expand the spectrum of CFTR pathogenic variations and can improve prenatal diagnosis and genetic counseling for cystic fibrosis.

Genetic Diagnosis of Pyruvate Kinase Deficiency in Undiagnosed Iranian Patients with Severe Hemolytic Anemia, using Whole Exome Sequencing.

After ruling out the most common causes of severe hemolytic anemia by routine diagnostic tests, certain patients remain without a diagnosis. The aim of this study was to elucidate the genetic cause of the disease in these patients using next generation sequencing (NGS). Four unrelated Iranian families including six blood transfusion dependent cases and their parents were referred to us from a specialist center in Tehran. There was no previous history of anemia in the families and the parents had no abnormal hematological presentations. All probands presented severe congenital hemolytic anemia, neonatal jaundice and splenomegaly. Common causes of hemolytic anemia were ruled out prior to this investigation in these patients and they had no diagnosis. Whole exome sequencing (WES) was performed in the probands and the results were confirmed by Sanger sequencing and subsequent family studies. We identified five variants in the PKLR gene, including a novel unpublished frameshift in these families. These variants were predicted as pathogenic according to the ACMG guidelines by Intervar and/or Varsome prediction tools. Subsequent family studies by Sanger sequencing supported the diagnosis of pyruvate kinase deficiency (PKD) in six affected individuals and the carrier status of disease in their parents. These findings show that PKD is among the rare blood disorders that could remain undiagnosed or even ruled out in Iranian population without performing NGS. This could be due to pitfalls in clinical, hematological or biochemical approaches in diagnosing PKD. Furthermore, genotyping PKD patients in Iran could reveal novel mutations in the PKLR gene.

Molecular diagnosis of maturity onset diabetes of the young in Iranian patients: improving management.

The purpose of this study is to identify the mutations of the most common form of maturity-onset diabetes of the young (MODY), also known as MODY3, in diabetic patients suspected of MODY. This can recommend appropriate medical surveillance of at-risk family members of MODY based on the genetic cause. We analyzed the clinical course of 19 patients from 12 unrelated Iranian families with diabetes features. The coding regions and intron-exon boundaries of the hepatocyte nuclear factor 1 alpha (HNF1A) gene were studied by polymerase chain reaction (PCR) and sanger sequencing. Also, the detected mutation was analyzed by bioinformatics tools. One novel frameshift insertion mutation (p.Glu11Argfs*12) was detected in one of the probands and seven other patients of her family with the heterozygote state. The mutation is located in the exon1 of the dimerization domain of the HNF1A gene. According to the In Silico analysis, the detected mutation is predicted as a pathogenic one. Differential diagnosis between MODY3 and other forms of diabetes can be considered a necessity in terms of overlapping symptoms of MODY3 with type1 or 2 diabetes. Molecular genetic testing can provide an accurate diagnosis for optimal management.

Prediction and interpretation of rare missense variant in OTOG associated with hearing loss

OTOG encodes for otogelin, a component of the tectorial membrane. This gene is associated with nonprogressive mild-to-moderate hearing loss. However, no studies have yet identified the association between OTOG variation and severe-to-profound hearing loss. Therefore, to address this issue, a family-based whole-exome sequencing strategy (WES) was carried out. Two unrelated Iranian families with non-syndromic hearing loss were identified, and WES was conducted on one selected candidate from each family. As a result, a rare homozygous missense variant, OTOG (c.C2383T:p.R795C), was detected in both of the subjected probands, and segregation analysis confirmed the c.C2383T variant in seven cases of severe-to-profound hearing loss. Additionally, the results from the protein modeling demonstrated that the altered position of a few disulfide bonds in the TIL domain may have a deleterious impact on protein stability and normal functionality. In conclusion, it seems that the homozygosity of the OTOG c.C2383T mutation sheds light on hearing loss pathobiology. Nevertheless, further studies are required to unravel the precise function of OTOG mutation, which is potentially associated with severe-to-profound hearing loss.

Novel variants in critical domains of ATP8A2 and expansion of clinical spectrum.

ATP8A2 is a P4-ATPase that flips phosphatidylserine across membranes to generate and maintain transmembrane phospholipid asymmetry. Loss-of-function variants cause severe neurodegenerative and developmental disorders. We have identified three ATP8A2 variants in unrelated Iranian families that cause intellectual disability, dystonia, below-average head circumference, mild optic atrophy, and developmental delay. Additionally, all the affected individuals displayed tooth abnormalities associated with defects in teeth development. Two variants (p.Asp825His and p.Met438Val) reside in critical functional domains of ATP8A2. These variants express at very low levels and lack ATPase activity. Inhibitor studies indicate that these variants are misfolded and degraded by the cellular proteasome. We conclude that Asp825, which coordinates with the Mg2+ ion within the ATP binding site, and Met438 are essential for the proper folding of ATP8A2 into a functional flippase. We also provide evidence on the association of tooth abnormalities with defects in ATP8A2, thereby expanding the clinical spectrum of the associated disease.

Identification of novel loss of function variants in MBOAT7 resulting in intellectual disability

The membrane bound O-acyltransferase domain-containing 7 (MBOAT7) gene codes for an enzyme involved in regulating arachidonic acid incorporation in lysophosphatidylinositol. Patients with homozygous nonsense mutations in MBOAT7 have intellectual disability (ID) accompanied with seizure and autism. Accumulating evidences obtained from human genetic studies have shown that MBOAT7 is also involved in fatty liver disease. Here we identified two novel homozygous variants in MBOAT7, NM_024298.5: c.1062C>A; p.(Tyr354*) and c.1135del; p.(Leu379Trpfs*9), in two unrelated Iranian families by means of whole exome sequencing. Sanger sequencing was performed to confirm the identified variants and also to investigate whether they co-segregate with the patients' phenotypes. To understand the functional consequences of these changes, we overexpressed recombinant wild type MBOAT7 and mutants in vitro and showed these mutations resulted in abolished protein synthesis and expression, indicating a complete loss of function. Albeit, we did not trace any liver diseases in our patients, but presence of globus pallidus signal changes in Magnetic Resonance Images might be indicative of metabolic changes as a result of loss of MBOAT7 expression in hepatic cells. These signal changes could also help as an important marker of MBOAT7 deficiency while analyzing the genomic data of patients with similar phenotypes.

Three Novel Variants identified in FBN1 and TGFBR2 in seven Iranian families with suspected Marfan syndrome.

BackgroundMarfan syndrome (MFS) is a multi‐systemic autosomal dominant disease of the connective tissue characterized by the early development of thoracic aneurysms/dissections, along with various manifestations of the ocular and skeletal systems. Due to the genetic and clinical heterogeneity, the clinical diagnosis of this disorder is challenging. Loss‐of‐function mutations in FBN1 (encodes fibrillin‐1) lead to MFS type 1. Also, similar mutations in transforming growth factor β receptor 2 (TGFBR2) gene cause MFS type 2. Both proteins involve in TGF‐β signaling.MethodsIn this study, genetic screening using a panel involving 14 genes, especially FBN1 and TGFBR2, were performed on seven representatives affected members of seven unrelated Iranian families suspected with MFS. To confirm the variants, Sanger sequencing was applied to other affected/unaffected members of the families.ResultsA total of 13 patients showed MFS manifestations. Using genetic screening, two novel and three previously reported variants in FBN1 were identified. We also detected two variants (a novel and a previously reported variant) in the TGFBR2 gene.ConclusionIn this study, we introduce three novel variants identified through gene screening in seven Iranian MFS families. This report is expected to considerably improve genetic counseling for Iranian MFS families. Early precise molecular diagnosis can be helpful for better management and improving the life expectancy of these patients.

Frequent Novel Variations Within MSH2 and MLH1 Genes in a Subset of Iranian Families With Hereditary Non-Polyposis Colorectal Cancer Shadi

Abstract- Hereditary non-polyposis colorectal cancer (HNPCC) is the most frequent autosomal dominant predisposition for development of colorectal cancer (CRC) caused by germline defects in mismatch repair (MMR) genes. Current study was aimed to find genetic variations in MSH2 and MLH1 genes and their correlation with the serum levels of Carcinoembryonic Antigen (CEA) in seven Iranian HNPCC families. Seven unrelated Iranian families including 11 HNPCC patients and 7 affected family members were selected. They were initially screened for mutations in exons 7 of MSH2 and exon 15 of MLH1 gene through polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP). Positive PCR results were further analyzed through exon sequencing. Serum CEA level was determined using the ELISA test. PCRSSCP was positive in 8 out of 18 patients (44%) for exons 7 of MSH2 gene, whereas two samples (11%) demonstrated to bear a mutation in exon 15 of the MLH1 gene. Sequencing analysis of both amplified exons in positive and negative samples have confirmed no mutation in negative samples while revealed 5 and 7 novel mutations in exons 7 and 15, respectively. The mean serum concentration of CEA had a significant difference between HNPCC patients and their healthy family members. Our results demonstrated that the PCR-SSCP method has high specificity and sensitivity in the first step of mutation screening of HNPCC families. High frequency of novel alterations found in the current assay may revise the mutation screening of MSH2 and MLH1 genes and abet further assessment of their frequency among individual HNPCC patients.

Genetic variability in Iranian limb-girdle muscular dystrophy type 2B patients: An evidence of a founder effect.

BackgroundDysferlinopathies are a group of autosomal recessive limb‐girdle muscular dystrophies (LGMDs) caused by mutations in DYSF (#603,009). This gene encodes a transmembrane protein called dysferlin. Since there are few reports on Iranian dysferlinopathy patients, we tried to identify the DYSF mutations in affected individuals of Iran.MethodsEight unrelated Iranian families have been selected for this study. Sanger sequencing followed by haplotype analysis was performed to identify individual variations in DYSF sequence. Identified variants were analyzed, and their pathogenicity was interpreted according to the recommendations of the American College of Medical Genetics and Genomics.ResultsWe identified two new mutations in DYSF, the first one is a nonsense mutation c.2419C > T (p.Gln807*), which eliminates downstream part of the protein. Another novel mutation is c. (1,053 + 1_1,054‐1)_(1,397 + 1_1,398‐1)del, which causes deletion of the DNA segment from exon 12 to exon 15.ConclusionTwo of the other six families are from the same ethnicity and share the same mutation and haplotype patterns, suggesting a founder mutation. Genetic analysis of dysferlinopathy can prevent a wrong diagnosis of myositis for these patients.

Identification of Two Novel Mutations in the ATM Gene from Patients with Ataxia-Telangiectasia by Whole Exome Sequencing.

Background Ataxia telangiectasia (AT) is one of the most common autosomal recessive hereditary ataxia presenting in childhood. The responsible gene for AT designated ATM (AT, mutated) encodes a protein which is involved in cell cycle checkpoints and other responses to genotoxicity. We describe two novel disease-causing mutations in two unrelated Iranian families with Ataxia-telangiectasia.Methods The probands including a 6-year-old female and an 18-year-old boy were diagnosed with Ataxia-telangiectasia among two different Iranian families. In this study, Whole-Exome Sequencing (WES) was employed for the detection of genetic changes in probands. The analysis of the co-segregation of the variants with the disease in families was conducted using PCR direct sequencing.Results Two novel frameshift mutations, (c.4236_4236del p. Pro1412fs) and (c.8907T>G p. Tyr2969Ter) in the ataxia telangiectasia mutated ATM gene were detected using Whole-Exome Sequencing (WES) in the probands. These mutations were observed in two separate A-T families.Conclusion Next-generation sequencing successfully identified the causative mutation in families with ataxia-telangiectasia. These novel mutations in the ATM gene reported in the present study could assist genetic counseling, Preimplantation Genetic Diagnosis (PGD) and prenatal diagnosis (PND) of AT.

Novel mutations in MYTH4-FERM domains of myosin 15 are associated with autosomal recessive nonsyndromic hearing loss

ObjectiveHereditary hearing loss is the most common neurosensory disorder in humans caused by myriad mutations in numerous genes. Autosomal recessive nonsyndromic hearing loss (ARNSHL) accounts for 80% of hearing impairments of genetic origin and is quite prevalent in societies with a high rate of consanguinity. In the current study, we investigated the causes of sensorineural hearing loss in 24 unrelated Iranian families who were mainly consanguineous and had at least two affected children. MethodsAll probands were initially screened for GJB2 mutations, as the most common causes of ARNSHL in Iran. Verified GJB2-negative samples were subsequently subjected to whole exome sequencing (WES) to identify the underlying causes of hearing impairment, and the variants identified in each family were further confirmed by Sanger sequencing. ResultsWES revealed three previously unreported mutations in MYO15A, the gene encoding the unconventional myosin 15 (Myo15). All variants identified, c.C6436T (p.R2146W), c.C9584G (p.P3195R) and c.G10266C (p.Q3422H), reside in the MYTH4 (myosin tail homology) and FERM (4.1 ezrin, radixin, moesin) domains of the protein. ConclusionGlobally, mutations in MYO15A are considered to be among the most prevalent genetic causes of ARNSHL, and they rank as the third leading cause of hearing loss in the Iranian population, below GJB2 and SLC26A4. Yet again, these results endorse the importance of MYO15 screening in hearing impaired populations, particularly in Iran.

A homozygous NOP14 variant is likely to cause recurrent pregnancy loss.

Recurrent pregnancy loss is newly defined as more than two consecutive miscarriages. Recurrent pregnancy loss occurs in <5% of total pregnancies. The cause in approximately 40-60% of recurrent pregnancy loss cases remains elusive and must be determined. We investigated two unrelated Iranian consanguineous families with recurrent pregnancy loss. We performed exome sequencing using DNA from a miscarriage tissue and identified a homozygous NOP14 missense variant (c.[136C>G];[136C>G]) in both families. NOP14 is an evolutionally conserved protein among eukaryotes and is required for 18S rRNA processing and 40S ribosome biogenesis. Interestingly, in zebrafish, homozygous mutation of nop14 (possibly loss of function) resulting from retrovirus-mediated insertional mutagenesis led to embryonic lethality at 5 days after fertilization, mimicking early pregnancy loss in humans. Similarly, it is known that the nop14-null yeast is inviable. These data suggest that the homozygous NOP14 mutation is likely to cause recurrent pregnancy loss. Furthermore, this study shows that exome sequencing is very useful to determine the etiology of unsolved recurrent pregnancy loss.

Identification of Three Novel Frameshift Mutations in the PKD1 Gene in Iranian Families with Autosomal Dominant Polycystic Kidney Disease Using Efficient Targeted Next-Generation Sequencing

Background/Aims: Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common inherited cystic kidney diseases caused by mutations in two large multi-exon genes, PKD1 and PKD2. High allelic heterogeneity and duplication of PKD1 exons 1–32 as six pseudo genes on chromosome 16 complicate molecular analysis of this disease. Methods: We applied targeted next-generation sequencing (NGS) in 9 non-consanguineous unrelated Iranian families with ADPKD to identify the genes hosting disease-causing mutations. This approach was confirmed by Sanger sequencing. Results: Here, we determined three different novel frameshift mutations and four previously reported nonsense mutations in the PKD1 gene encoding polycystin1 in heterozygotes. Conclusion: This study demonstrates the effectiveness of NGS in significantly reducing the cost and time for simultaneous sequence analysis of PKD1 and PKD2, simplifying the genetic diagnostics of ADPKD. Although a probable correlation between the mutation types and phenotypic outcome is possible, however for more extensive studies in future, the consideration of renal hypouricemia (RHUC) and PKD1 coexistence may be helpful. The novel frameshift mutations reported by this study are p. Q1997X, P. D73X and p. V336X.