Rituximab in combination with cyclosporine and steroid pulse therapy for childhood-onset multidrug-resistant nephrotic syndrome: a multicenter single-arm clinical trial (JSKDC11 trial).

BACKGROUND: Only 80% of children with idiopathic nephrotic syndrome respond well to glucocorticoid therapy. Multidrug-resistant nephrotic syndrome (MRNS) is associated with a poor kidney prognosis. Several retrospective studies have identified rituximab as an effective treatment for MRNS; however, prospective studies are required to assess its efficacy and safety. METHODS: We conducted a multicenter, non-blinded, single-arm trial to investigate the efficacy and safety of rituximab in patients with childhood-onset MRNS who were resistant to cyclosporine and more than three courses of steroid pulse therapy. The enrolled patients received four 375 mg/m2 doses of rituximab in combination with baseline cyclosporine and steroid pulse therapy. The primary endpoint was a > 50% reduction in the urinary protein/creatinine ratio from baseline on day 169. Complete and partial remissions were also evaluated. RESULTS: Six patients with childhood-onset MRNS were enrolled. All patients were negative for pathogenic variants of podocyte-related genes. On day 169, five patients (83.3%) showed a > 50% reduction in the urinary protein/creatinine ratio, two patients showed partial remission, and two patients showed complete remission. No deaths occurred and severe adverse events occurred in two patients (infection in one patient and acute kidney injury in one patient). Three patients needed treatment for moderate-to-severe infection. CONCLUSIONS: The study treatment effectively reduced the urinary protein/creatinine ratio in patients with childhood-onset MRNS. The adverse events in this study were within the expected range; however, attention should be paid to the occurrence of infections.

Aberrant hypomethylation at imprinted differentially methylated regions is involved in biparental placental mesenchymal dysplasia.

BACKGROUND: Placental mesenchymal dysplasia (PMD) is a morphological abnormality resembling partial hydatidiform moles. It is often associated with androgenetic/biparental mosaicism (ABM) and complicated by Beckwith-Wiedemann syndrome (BWS), an imprinting disorder. These phenomena suggest an association between PMD and aberrant genomic imprinting, particularly of CDKN1C and IGF2. The existence of another type of PMD containing the biparental genome has been reported. However, the frequency and etiology of biparental PMD are not yet fully understood. RESULTS: We examined 44 placental specimens from 26 patients with PMD: 19 of these were macroscopically normal and 25 exhibited macroscopic PMD. Genotyping by DNA microarray or short tandem repeat analysis revealed that approximately 35% of the macroscopic PMD specimens could be classified as biparental, while the remainder were ABM. We performed a DNA methylation analysis using bisulfite pyrosequencing of 15 placenta-specific imprinted differentially methylated regions (DMRs) and 36 ubiquitous imprinted DMRs. As expected, most DMRs in the macroscopic PMD specimens with ABM exhibited the paternal epigenotype. Importantly, the biparental macroscopic PMD specimens exhibited frequent aberrant hypomethylation at seven of the placenta-specific DMRs. Allelic expression analysis using single-nucleotide polymorphisms revealed that five imprinted genes associated with these aberrantly hypomethylated DMRs were biallelically expressed. Frequent aberrant hypomethylation was observed at five ubiquitous DMRs, including GRB10 but not ICR2 or ICR1, which regulate the expression of CDKN1C and IGF2, respectively. Whole-exome sequencing performed on four biparental macroscopic PMD specimens did not reveal any pathological genetic abnormalities. Clinical and molecular analyses of babies born from pregnancies with PMD revealed four cases with BWS, each exhibiting different molecular characteristics, and those between BWS and PMD specimens were not always the same. CONCLUSION: These data clarify the prevalence of biparental PMD and ABM-PMD and strongly implicate hypomethylation of DMRs in the pathogenesis of biparental PMD, particularly placenta-specific DMRs and the ubiquitous GRB10, but not ICR2 or ICR1. Aberrant hypomethylation of DMRs was partial, indicating that it occurs after fertilization. PMD is an imprinting disorder, and it may be a missing link between imprinting disorders and placental disorders incompatible with life, such as complete hydatidiform moles and partial hydatidiform moles.

Identification of novel OCRL isoforms associated with phenotypic differences between Dent disease-2 and Lowe syndrome.

BACKGROUND: Although Lowe syndrome and Dent disease-2 are caused by Oculocerebrorenal syndrome of Lowe (OCRL) mutations, their clinical severities differ substantially and their molecular mechanisms remain unclear. Truncating mutations in OCRL exons 1-7 lead to Dent disease-2, whereas those in exons 8-24 lead to Lowe syndrome. Herein we identified the mechanism underlying the action of novel OCRL protein isoforms. METHODS: Messenger RNA samples extracted from cultured urine-derived cells from a healthy control and a Dent disease-2 patient were examined to detect the 5' end of the OCRL isoform. For protein expression and functional analysis, vectors containing the full-length OCRL transcripts, the isoform transcripts and transcripts with truncating mutations detected in Lowe syndrome and Dent disease-2 patients were transfected into HeLa cells. RESULTS: We successfully cloned the novel isoform transcripts from OCRL exons 6-24, including the translation-initiation codons present in exon 8. In vitro protein-expression analysis detected proteins of two different sizes (105 and 80 kDa) translated from full-length OCRL, whereas only one protein (80 kDa) was found from the isoform and Dent disease-2 variants. No protein expression was observed for the Lowe syndrome variants. The isoform enzyme activity was equivalent to that of full-length OCRL; the Dent disease-2 variants retained >50% enzyme activity, whereas the Lowe syndrome variants retained <20% activity. CONCLUSIONS: We elucidated the molecular mechanism underlying the two different phenotypes in OCRL-related diseases; the functional OCRL isoform translated starting at exon 8 was associated with this mechanism.

Mycophenolate Mofetil after Rituximab for Childhood-Onset Complicated Frequently-Relapsing or Steroid-Dependent Nephrotic Syndrome.

BACKGROUND: Rituximab is the standard therapy for childhood-onset complicated frequently relapsing or steroid-dependent nephrotic syndrome (FRNS/SDNS). However, most patients redevelop FRNS/SDNS after peripheral B cell recovery. METHODS: We conducted a multicenter, randomized, double-blind, placebo-controlled trial to examine whether mycophenolate mofetil (MMF) administration after rituximab can prevent treatment failure (FRNS, SDNS, steroid resistance, or use of immunosuppressive agents or rituximab). In total, 39 patients (per group) were treated with rituximab, followed by either MMF or placebo until day 505 (treatment period). The primary outcome was time to treatment failure (TTF) throughout the treatment and follow-up periods (until day 505 for the last enrolled patient). RESULTS: TTFs were clinically but not statistically significantly longer among patients given MMF after rituximab than among patients receiving rituximab monotherapy (median, 784.0 versus 472.5 days, hazard ratio [HR], 0.59; 95% confidence interval [95% CI], 0.34 to 1.05, log-rank test: P=0.07). Because most patients in the MMF group presented with treatment failure after MMF discontinuation, we performed a post-hoc analysis limited to the treatment period and found that MMF after rituximab prolonged the TTF and decreased the risk of treatment failure by 80% (HR, 0.20; 95% CI, 0.08 to 0.50). Moreover, MMF after rituximab reduced the relapse rate and daily steroid dose during the treatment period by 74% and 57%, respectively. The frequency and severity of adverse events were similar in both groups. CONCLUSIONS: Administration of MMF after rituximab may sufficiently prevent the development of treatment failure and is well tolerated, although the relapse-preventing effect disappears after MMF discontinuation.

Early Elevation of Complement Factor Ba Is a Predictive Biomarker for Transplant-Associated Thrombotic Microangiopathy.

Transplant-associated thrombotic microangiopathy (TA-TMA) is a fatal complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Previous reports suggest that TA-TMA is caused by complement activation by complement-related genetic variants; however, this needs to be verified, especially in adults. Here, we performed a nested case-control study of allo-HSCT-treated adults at a single center. Fifteen TA-TMA patients and 15 non-TA-TMA patients, matched according to the propensity score, were enrolled. Based on a previous report showing an association between complement-related genes and development of TA-TMA, we first sequenced these 17 genes. Both cohorts harbored several genetic variants with rare allele frequencies; however, there was no difference in the percentage of patients in the TA-TMA and non-TA-TMA groups with the rare variants, or in the average number of rare variants per patient. Second, we measured plasma concentrations of complement proteins. Notably, levels of Ba protein on Day 7 following allo-HSCT were abnormally and significantly higher in TA-TMA than in non-TA-TMA cases, suggesting that complement activation via the alternative pathway contributes to TA-TMA. All other parameters, including soluble C5b-9, on Day 7 were similar between the groups. The levels of C3, C4, CH50, and complement factors H and I in the TA-TMA group after Day 28 were significantly lower than those in the non-TA-TMA group. Complement-related genetic variants did not predict TA-TMA development. By contrast, abnormally high levels of Ba on Day 7 did predict development of TA-TMA and non-relapse mortality. Thus, Ba levels on Day 7 after allo-HSCT are a sensitive and prognostic biomarker of TA-TMA.

Clinical Features, Molecular Genetics, and Long-Term Outcome in Congenital Chloride Diarrhea: A Nationwide Study in Japan.

OBJECTIVE: To clarify clinical and genetic features of Japanese children with congenital chloride diarrhea (CCD). STUDY DESIGN: This was a multi-institutional, retrospective survey of 616 pediatric centers in Japan with identified patients with CCD between 2014 and 2018. Mutations involving SLC26A3 were detected by Sanger sequencing. RESULTS: Thirteen patients met all entry criteria including mutations in SLC26A3, and 14 patients satisfied clinical diagnostic criteria. Homozygous or compound heterozygous mutations in SLC26A3, including 6 novel mutations, were identified in 13 of these 14 patients (93%). The most common (detected in 7 of 13) was c.2063-1g>t. Median age at diagnosis was 1 day. Nine of the patients meeting all criteria were diagnosed as neonates (69%). Median follow-up duration was 10 years. When studied, 8 patients had <5 stools daily (62%), and all had fewer than in infancy. Only 1 patient had nephrocalcinosis, and 3 (23%) had mild chronic kidney disease. Neurodevelopment was generally good; only 1 patient required special education. Five patients (38%) received long-term sodium, potassium, and chloride supplementation. CONCLUSIONS: Early fetal ultrasound diagnosis and prompt long-term sodium, potassium, and chloride supplementation were common management features. Genetic analysis of SLC26A3 provided definitive diagnosis of CCD. In contrast with previously reported localities, c.2063-1g>t might be a founder mutation in East Asia.

Association between Crohn's disease and AarF domain-containing kinase 4 glomerulopathy.

Coenzyme Q10 deficiency causing gastrointestinal symptoms has not been reported. At least 15 genes are involved in CoQ10 biosynthesis, and one of the genes is AarF domain-containing kinase 4 (ADCK4). This case report first showed a patient who presented with Crohn's disease (CD) combined with ADCK4 glomerulopathy (ADCK4-GN). After approximately 2 years of infliximab treatment for CD, this patient has remained in clinical remission with no adverse effects. Moreover, two important findings were obtained: first, individuals with ADCK4-GN may present with complications, such as CD, which is a gastrointestinal disease, and infliximab may be effective for CD associated with ADCK4-GN. Second, infliximab may also be effective and safe for individuals with CD who are undergoing peritoneal dialysis. Thus, the results of the present study may be used in managing patients with such condition, and as a result, better clinical outcomes will be obtained.

Atypical Hemolytic Uremic Syndrome With the p.Ile1157Thr C3 Mutation Successfully Treated With Plasma Exchange and Eculizumab: A Case Report.

To describe a case of atypical hemolytic uremic syndrome induced by influenza A infection with the p.Ile1157Thr C3 mutation. DATA SOURCES: Clinical observations of a patient. STUDY SELECTION: Case reports. DATA EXTRACTION: Data extracted from medical records, after patient's consent. DATA SYNTHESIS: Four days prior to presentation to our hospital, a 16-year-old adolescent had a fever and arthralgia with hematuria. He was found to be positive for type A influenza and prescribed oseltamivir and acetaminophen by a primary-care physician. A bleeding tendency and purpura in the extremities and on the trunk developed; therefore, he was transferred to Chiba University Hospital. Hematology revealed severe thrombocytopenia, hyperbilirubinemia, and acute kidney injury. Aspartate aminotransferase, lactate dehydrogenase, and potassium could not be determined because of severe hemolysis. Highly elevated blood urea nitrogen and creatinine levels indicated acute kidney injury. A platelet count of 24,000/µL indicated thrombocytopenia, with low hemoglobin level. Peripheral blood profiling identified schistocytes. Continuous hemodiafiltration and plasma infusion were initiated immediately; however, he became oliguric. Plasma exchange was initiated on ICU day 3, but decreased urine output, hemolysis, and thrombocytopenia persisted. IV eculizumab therapy was initiated on day 7 and resulted in recovery of these symptoms and also successful discontinuation of renal support. The patient showed a stable condition without recurrence of hemolytic findings and acute kidney injury and is currently on maintenance therapy of eculizumab (1,200 mg, every other week) without any relapse of atypical hemolytic uremic syndrome symptoms. A plasma sample collected prior to initiation of plasma exchange showed an disintegrin-like and metalloprotease with thrombospondin type 1 motifs 13 activity level of 104.9%. The absence of both Shiga toxin-producing Escherichia coli in feces led to suspicion of atypical hemolytic uremic syndrome. Subsequent genetic analysis identified a mutation in C3 (p.Ile1157Thr), confirming the diagnosis of atypical hemolytic uremic syndrome. CONCLUSIONS: Although managing thrombocytopenia secondary to infection, inclusion of atypical hemolytic uremic syndrome in the differential diagnosis at an early stage is important in clinical practice.

Strong Association of the HLA-DR/DQ Locus with Childhood Steroid-Sensitive Nephrotic Syndrome in the Japanese Population.

Background Nephrotic syndrome is the most common cause of chronic glomerular disease in children. Most of these patients develop steroid-sensitive nephrotic syndrome (SSNS), but the loci conferring susceptibility to childhood SSNS are mainly unknown.Methods We conducted a genome-wide association study (GWAS) in the Japanese population; 224 patients with childhood SSNS and 419 adult healthy controls were genotyped using the Affymetrix Japonica Array in the discovery stage. Imputation for six HLA genes (HLA-A, -C, -B, -DRB1, -DQB1, and -DPB1) was conducted on the basis of Japanese-specific references. We performed genotyping for HLA-DRB1/-DQB1 using a sequence-specific oligonucleotide-probing method on a Luminex platform. Whole-genome imputation was conducted using a phased reference panel of 2049 healthy Japanese individuals. Replication was performed in an independent Japanese sample set including 216 patients and 719 healthy controls. We genotyped candidate single-nucleotide polymorphisms using the DigiTag2 assay.Results The most significant association was detected in the HLA-DR/DQ region and replicated (rs4642516 [minor allele G], combined Pallelic=7.84×10-23; odds ratio [OR], 0.33; 95% confidence interval [95% CI], 0.26 to 0.41; rs3134996 [minor allele A], combined Pallelic=1.72×10-25; OR, 0.29; 95% CI, 0.23 to 0.37). HLA-DRB1*08:02 (Pc=1.82×10-9; OR, 2.62; 95% CI, 1.94 to 3.54) and HLA-DQB1*06:04 (Pc=2.09×10-12; OR, 0.10; 95% CI, 0.05 to 0.21) were considered primary HLA alleles associated with childhood SSNS. HLA-DRB1*08:02-DQB1*03:02 (Pc=7.01×10-11; OR, 3.60; 95% CI, 2.46 to 5.29) was identified as the most significant genetic susceptibility factor.Conclusions The most significant association with childhood SSNS was detected in the HLA-DR/DQ region. Further HLA allele/haplotype analyses should enhance our understanding of molecular mechanisms underlying SSNS.

Clinically diverse phenotypes and genotypes of patients with branchio-oto-renal syndrome.

Branchio-oto-renal (BOR) syndrome is a rare autosomal dominant disorder characterized by branchiogenic anomalies, hearing loss, and renal anomalies. The aim of this study was to reveal the clinical phenotypes and their causative genes in Japanese BOR patients. Patients clinically diagnosed with BOR syndrome were analyzed by direct sequencing, multiplex ligation-dependent probe amplification (MLPA), array-based comparative genomic hybridization (aCGH), and next-generation sequencing (NGS). We identified the causative genes in 38/51 patients from 26/36 families; EYA1 aberrations were identified in 22 families, SALL1 mutations were identified in two families, and SIX1 mutations and a 22q partial tetrasomy were identified in one family each. All patients identified with causative genes suffered from hearing loss. Second branchial arch anomalies, including a cervical fistula or cyst, preauricular pits, and renal anomalies, were frequently identified (>60%) in patients with EYA1 aberrations. Renal hypodysplasia or unknown-cause renal insufficiency was identified in more than half of patients with EYA1 aberrations. Even within the same family, renal phenotypes often varied substantially. In addition to direct sequencing, MLPA and NGS were useful for the genetic analysis of BOR patients.

Successful Treatment of Transplantation-associated Atypical Hemolytic Uremic Syndrome With Eculizumab.

We herein reported a 4-month-old boy with transplantation-associated atypical hemolytic uremic syndrome (TA-aHUS) who was successfully treated with eculizumab. The patient diagnosed with type 3 of familial hemophagocytic lymphohistiocytosis underwent cord blood transplantation. After transplantation, he developed TA-aHUS, but plasma exchanges were unsuccessful. We identified deletions in CFH-related gene 1 (del-CFHR1) by the multiplex ligation-dependent probe amplification testing procedure and CFH autoantibodies. Eculizumab has been administered to the patient, with a marked improvement being achieved in thrombocytopenia. He has been well except for the persistent microhematuria for a year after transplantation. Uncontrolled complement activation might be involved in the pathophysiology of TA-aHUS.

Postoperative Atypical Hemolytic Uremic Syndrome Treated Successfully with Eculizumab.

Atypical hemolytic uremic syndrome (aHUS) is a rare type of HUS associated with dysregulation of the alternative complement pathway. We herein report the findings of a 43-year-old woman who developed aHUS two days after myomectomy. The serum C3 level was low and the sheep erythrocyte hemolytic assay showed a moderate hemolysis. The renal insufficiency progressed despite performing plasma exchange therapy, although a partial hematological response was observed. Thus, the patient was subsequently treated with the anti-C5 antibody eculizumab, which promptly improved and normalized the renal function. Although aHUS after surgery has rarely been reported, it needs to be promptly recognized and treated with eculizumab to prevent irreversible renal damage.

Identification of consensus motifs associated with mitotic recombination and clinical characteristics in patients with paternal uniparental isodisomy of chromosome 11.

Uniparental disomy (UPD) is defined as the inheritance of both homologs of a given genomic region from only one parent. The majority of UPD includes an entire chromosome. However, the extent of UPD is sometimes limited to a subchromosomal region (segmental UPD). Mosaic paternal UPD (pUPD) of chromosome 11 is found in approximately 20% of patients with Beckwith-Wiedemann syndrome (BWS) and almost all pUPDs are segmental isodisomic pUPDs resulting from mitotic recombination at an early embryonic stage. A mechanism initiating a DNA double strand break (DSB) within 11p has been predicted to lead to segmental pUPD. However, no consensus motif has yet been found. Here, we analyzed 32 BWS patients with pUPD by SNP array and searched for consensus motifs. We identified four consensus motifs frequently appearing within breakpoint regions of segmental pUPD. These motifs were found in another nine BWS patients with pUPD. In addition, the seven motifs found in meiotic recombination hot spots could not be found within pUPD breakpoint regions. Histone H3 lysine 4 trimethylation, a marker of DSB initiation, could not be found either. These findings suggest that the mechanism(s) of mitotic recombination leading to segmental pUPD are different from that of meiotic recombination. Furthermore, we found seven patients with paternal uniparental diploidy (PUD) mosaicism. Comparison of clinical features between segmental pUPDs and PUDs showed that developmental disability and cardiac abnormalities were additional characteristic features of PUD mosaicism, along with high risk of tumor development. We also found that macroglossia was characteristic of segmental pUPD mosaicism.

Alport syndrome caused by a COL4A5 deletion and exonization of an adjacent AluY.

Mutation-induced activation of splice sites in intronic repetitive sequences has contributed significantly to the evolution of exon-intron structure and genetic disease. Such events have been associated with mutations within transposable elements, most frequently in mutation hot-spots of Alus. Here, we report a case of Alu exonization resulting from a 367-nt genomic COL4A5 deletion that did not encompass any recognizable transposed element, leading to the Alport syndrome. The deletion brought to proximity the 5' splice site of COL4A5 exon 33 and a cryptic 3' splice site in an antisense AluY copy in intron 32. The fusion exon was depleted of purines and purine-rich splicing enhancers, but had low levels of intramolecular secondary structure, was flanked by short introns and had strong 5' and Alu-derived 3' splice sites, apparently compensating poor composition and context of the new exon. This case demonstrates that Alu splice sites can be activated by outlying deletions, highlighting Alu versatility in shaping the exon-intron organization and expanding the spectrum of mutational mechanisms that introduce repetitive sequences in mRNAs.

Comprehensive and quantitative multilocus methylation analysis reveals the susceptibility of specific imprinted differentially methylated regions to aberrant methylation in Beckwith-Wiedemann syndrome with epimutations.

PURPOSE: Expression of imprinted genes is regulated by DNA methylation of differentially methylated regions (DMRs). Beckwith-Wiedemann syndrome is an imprinting disorder caused by epimutations of DMRs at 11p15.5. To date, multiple methylation defects have been reported in Beckwith-Wiedemann syndrome patients with epimutations; however, limited numbers of DMRs have been analyzed. The susceptibility of DMRs to aberrant methylation, alteration of gene expression due to aberrant methylation, and causative factors for multiple methylation defects remain undetermined. METHODS: Comprehensive methylation analysis with two quantitative methods, matrix-assisted laser desorption/ionization mass spectrometry and bisulfite pyrosequencing, was conducted across 29 DMRs in 54 Beckwith-Wiedemann syndrome patients with epimutations. Allelic expressions of three genes with aberrant methylation were analyzed. All DMRs with aberrant methylation were sequenced. RESULTS: Thirty-four percent of KvDMR1-loss of methylation patients and 30% of H19DMR-gain of methylation patients showed multiple methylation defects. Maternally methylated DMRs were susceptible to aberrant hypomethylation in KvDMR1-loss of methylation patients. Biallelic expression of the genes was associated with aberrant methylation. Cis-acting pathological variations were not found in any aberrantly methylated DMR. CONCLUSION: Maternally methylated DMRs may be vulnerable to DNA demethylation during the preimplantation stage, when hypomethylation of KvDMR1 occurs, and aberrant methylation of DMRs affects imprinted gene expression. Cis-acting variations of the DMRs are not involved in the multiple methylation defects.

Homozygous deletion of DIS3L2 exon 9 due to non-allelic homologous recombination between LINE-1s in a Japanese patient with Perlman syndrome.

Perlman syndrome is a rare, autosomal recessive overgrowth disorder. Recently, the deletion of exon 9 and other mutations of the DIS3L2 gene have been reported in patients; however, the mechanism behind this deletion is still unknown. We report the homozygous deletion of exon 9 of DIS3L2 in a Japanese patient with Perlman syndrome. We identified the deletion junction, and implicate a non-allelic homologous recombination (NAHR) between two LINE-1 (L1) elements as the causative mechanism. Furthermore, the deletion junctions were different between the paternal and maternal mutant alleles, suggesting the occurrence of two independent NAHR events in the ancestors of each parent. The data suggest that the region around exon 9 might be a hot spot of L1-mediated NAHR.

Detection of large deletion mutations in the COL4A5 gene of female Alport syndrome patients.

Alport syndrome is the most common form of hereditary nephritis, and the majority of cases are caused by mutations in the COL4A5 gene. However, direct sequencing by polymerase chain reaction (PCR), from genomic DNA, or reverse transcriptase-polymerase chain reaction (RT-PCR), from mRNA, or polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) has reportedly resulted in detection rates of 31% to 84%, but of only 20% to 71% when restricted to female patients. This report concerns two female patients with X-linked Alport syndrome. Although mutational analysis of the COL4A5 gene was conducted with direct sequencing using genomic DNA and mRNA extracted from leukocytes, the results were negative for detection of mutations. Semi-quantitative PCR using genomic DNA was therefore conducted to detect large heterozygous deletions. The results were that the first patient showed complete loss of the COL4A5 gene and the second patient showed deletion from exons 37 to 51. Our patients possessed large heterozygous deletions in the COL4A5 gene that could not be detected with the standard direct sequencing method and were identified with semi-quantitative PCR. Previously reported mutation detection rates for female patients have been lower than overall rates. Our findings indicate that this difference may, in part, be due to failure to detect this type of mutation with conventional analytical methods.