Novel dominant KATP channel mutations in infants with congenital hyperinsulinism: Validation by in vitro expression studies and in vivo carrier phenotyping

Inactivating mutations in the genes encoding the two subunits of the pancreatic beta‐cell K_ATP channel, ABCC8 and KCNJ11, are the most common finding in children with congenital hyperinsulinism (HI). Interpreting novel missense variants in these genes is problematic, because they can be either dominant or recessive mutations, benign polymorphisms, or diabetes mutations. This report describes six novel missense variants in ABCC8 and KCNJ11 that were identified in 11 probands with congenital HI. One of the three ABCC8 mutations (p.Ala1458Thr) and all three KCNJ11 mutations were associated with responsiveness to diazoxide. Sixteen family members carried the ABCC8 or KCNJ11 mutations; only two had hypoglycemia detected at birth and four others reported symptoms of hypoglycemia. Phenotype testing of seven adult mutation carriers revealed abnormal protein‐induced hypoglycemia in all; fasting hypoketotic hypoglycemia was demonstrated in four of the seven. All of six mutations were confirmed to cause dominant pathogenic defects based on in vitro expression studies in COSm6 cells demonstrating normal trafficking, but reduced responses to MgADP and diazoxide. These results indicate a combination of in vitro and in vivo phenotype tests can be used to differentiate dominant from recessive K_ATP channel HI mutations and personalize management of children with congenital HI.     

Carrier screening in the era of expanding genetic technology

PurposeThe Center for Jewish Genetics provides genetic education and carrier screening to individuals of Jewish descent. Carrier screening has traditionally been performed by targeted mutation analysis for founder mutations with an enzyme assay for Tay–Sachs carrier detection. The development of next-generation sequencing (NGS) allows for higher detection rates regardless of ethnicity. Here, we explore differences in carrier detection rates between genotyping and NGS in a primarily Jewish population.MethodsPeripheral blood samples or saliva samples were obtained from 506 individuals. All samples were analyzed by sequencing, targeted genotyping, triplet-repeat detection, and copy-number analysis; the analyses were carried out at Counsyl.ResultsOf 506 individuals screened, 288 were identified as carriers of at least 1 condition and 8 couples were carriers for the same disorder. A total of 434 pathogenic variants were identified. Three hundred twelve variants would have been detected via genotyping alone. Although no additional mutations were detected by NGS in diseases routinely screened for in the Ashkenazi Jewish population, 26.5% of carrier results and 2 carrier couples would have been missed without NGS in the larger panel.ConclusionIn a primarily Jewish population, NGS reveals a larger number of pathogenic variants and provides individuals with valuable information for family planning.Genet Med 18 12, 1214–1217.     

The spectrum of ABCC8 mutations in Norwegian patients with congenital hyperinsulinism of infancy

Potassium channels in the plasma membrane of the pancreatic beta cells are critical in maintaining glucose homeostasis by responding to ATP and coupling metabolic changes to insulin secretion. These channels consist of subunits denoted the sulfonylurea receptor SUR1 and the inwardly rectifying ion channel KIR6.2, which are encoded by the genes ABCC8 and KCNJ11 , respectively. Activating mutations in the subunit genes can result in monogenic diabetes, whereas inactivating mutations are the most common cause of congenital hyperinsulinism of infancy (CHI). Twenty-six Norwegian probands with CHI were analyzed for alterations in ABCC8 and KCNJ11 . Fifteen probands (58%) had mutations in the ABCC8 gene. Nine patients were homozygous or compound heterozygous for the mutations, indicating diffuse pancreatic disease. In five patients, heterozygous and paternally inherited mutations were found, suggesting focal disease. One patient had a de novo mutation likely to cause a milder, dominant form of CHI. Altogether, 16 different ABCC8 mutations (including the novel alterations W231R, C267X, IVS6-3C>G, I462V, Q917X and T1531A) were identified. The mutations IVS10+1G>T, R1493W and V21D occurred in five, three and two families, respectively. KCNJ11 mutations were not found in any patients. Based on our mutation screening, we estimate the minimum birth prevalence of ABCC8 -CHI in Norway to 1:70,000 during the past decade. Our results considerably extend the knowledge of the molecular genetics behind CHI in Scandinavia.     

Carrier frequency of two BBS2 mutations in the Ashkenazi population

Bardet–Biedl syndrome (BBS) is known to be caused by numerous mutations that occur in at least 15 of the BBS genes. As the disease follows an autosomal recessive pattern of inheritance, carrier screening can be performed for at‐risk couples, but the number of potential mutation sites to screen can be daunting. Ethnic studies can help to narrow this range by highlighting mutations that are present at higher percentages in certain populations. In this article, the carrier frequency for two mutations that occur in the BBS2 gene, c.311A>C and c.1895G>C were studied in individuals of Ashkenazi Jewish descent in order to advise on including them in existing mutation panels for this population. Carrier screenings were performed on individuals from the Ashkenazi Jewish population using a combination of TaqMan genotyping assays followed by real‐time polymerase chain reaction (PCR) and allelic discrimination, and allele‐specific PCR confirmed by restriction analysis. The combined results indicated carrier frequencies of 0.473% (±0.0071%) for the c.311A>C mutation and 0.261% (±0.0064%) for the c.1895G>C mutation. On the basis of these frequencies, we believe that the two mutations should be considered for inclusion in screening panels for the Ashkenazi population.     

Update of variants identified in the pancreatic β‐cell KATP channel genes KCNJ11 and ABCC8 in individuals with congenital hyperinsulinism and diabetes

The most common genetic cause of neonatal diabetes and hyperinsulinism is pathogenic variants in ABCC8 and KCNJ11. These genes encode the subunits of the β‐cell ATP‐sensitive potassium channel, a key component of the glucose‐stimulated insulin secretion pathway. Mutations in the two genes cause dysregulated insulin secretion; inactivating mutations cause an oversecretion of insulin, leading to congenital hyperinsulinism, whereas activating mutations cause the opposing phenotype, diabetes. This review focuses on variants identified in ABCC8 and KCNJ11, the phenotypic spectrum and the treatment implications for individuals with pathogenic variants.     

A deleterious mutation in the PEX2 gene causes Zellweger syndrome in individuals of Ashkenazi Jewish descent

Zellweger syndrome is known to be caused by numerous mutations that occur in at least 12 of the PEX genes. While phenotypes vary, many are severely debilitating, and death can result in affected newborns. Since the disease follows an autosomal recessive pattern of inheritance, carrier screening can be done for at‐risk couples, but the number of potential mutations sites to screen can be daunting. Ethnicity‐specific studies can help narrow this range by highlighting mutations that are present at higher percentages in certain populations. In this article, the carrier frequencies for two mutations causative of the severe Zellweger syndrome spectrum phenotype that occur in the PEX2 gene, c.355C>T and c.550del, were studied in individuals of Ashkenazi Jewish descent in order to advise on inclusion in existing carrier screening mutation panels for this population. The screening was performed for 2093 individuals through the use of TaqMan genotyping assays, real‐time PCR, and allelic discrimination. Results indicated a carrier frequency of 0.813% (±0.385%) for the c.355C>T mutation and a carrier frequency of 0.00% (±0.00%) for the c.550del mutation. On the basis of these frequencies, we believe that the c.355C>T mutation should be considered for inclusion in carrier screening panels for the Ashkenazi population.     

Identification of two novel frameshift mutations in the KCNJ11 gene in two Italian patients affected by Congenital Hyperinsulinism of Infancy

Congenital Hyperinsulinism of Infancy (CHI) is a genetically heterogeneous disorder characterized by profound hypoglycemia related to inappropriate insulin secretion. Two histopathologically and genetically distinct groups are recognized among patients with CHI due to ATP-sensitive potassium channel (KATP) defects: a diffuse type (Di-CHI), which involves the whole pancreas, and a focal form (Fo-CHI), which shows adenomatous islet-cell hyperplasia of a particular area within the normal pancreas. The beta-cell KATP channel consists of two essential subunits: Kir6.2 encoded by the KCNJ11 gene which is the pore-forming unit and belongs to the inwardly rectifying potassium channel family, and SUR1 (sulfonylurea receptor 1) encoded by the ABCC8 gene, which belongs to the ATP-binding cassette (ABC) transporter family. The KATP channel is an octameric complex of four Kir6.2 and four SUR1 subunits. More than one hundred mutations have been found in KATP channel genes ABCC8 and KCNJ11, but to date only twenty mutations have been identified in KCNJ11, most of them are missense mutations and only one is a single base deletion. The Fo-CHI has been demonstrated to arise in individuals who have a germline mutation in the paternal allele of ABCC8 or KCNJ11 in addition to a somatic loss of the maternally derived chromosome region 11p15 in adenomatous pancreatic beta-cells, while Di-CHI predominantly arises from the autosomal recessive inheritance of KATP channel gene mutations. Here we describe the molecular findings in nine children who presented, in the neonatal period, with signs and symptoms of hypoglycemia and diagnosed affected by CHI according to international diagnostic criteria. Direct sequencing of the complete coding exon and promoter region of KCNJ11 gene showed, in two Italian patients, two new heterozygous mutations which result in the appearance of premature translation termination codons resulting in the premature end of Kir6.2. Interestingly most of the CHI mutations detected in other population studies are situated in the ABCC8 gene.     

Mutation of KCNJ8 in a patient with Cantú syndrome with unique vascular abnormalities – Support for the role of K(ATP) channels in this condition

KCNJ8 (NM_004982) encodes the pore forming subunit of one of the ATP-sensitive inwardly rectifying potassium (K_ATP) channels. KCNJ8 sequence variations are traditionally associated with J-wave syndromes, involving ventricular fibrillation and sudden cardiac death. Recently, the K_ATP gene ABCC9 (SUR2, NM_020297) has been associated with the multi-organ disorder Cantú syndrome or hypertrichotic osteochondrodysplasia (MIM 239850) (hypertrichosis, macrosomia, osteochondrodysplasia, and cardiomegaly). Here, we report on a patient with a de novo nonsynonymous KCNJ8 SNV (p.V65M) and Cantú syndrome, who tested negative for mutations in ABCC9. The genotype and multi-organ abnormalities of this patient are reviewed. A careful screening of the K_ATP genes should be performed in all individuals diagnosed with Cantú syndrome and no mutation in ABCC9.     

Identification of the first non-Jewish mutation in familial Dysautonomia

Familial Dysautonomia is an autosomal recessive disease with a remarkably high carrier frequency in the Ashkenazi Jewish population. It has recently been estimated that as many as 1 in 27 Ashkenazi Jews is a carrier of FD. The FD gene has been identified as IKBKAP, and two disease-causing mutations have been identified. The most common mutation, which is present on 99.5% of all FD chromosomes, is an intronic splice site mutation that results in tissue-specific skipping of exon 20. The second mutation, R696P, is a missense mutation that has been identified in 4 unrelated patients heterozygous for the major splice mutation. Interestingly, despite the fact that FD is a recessive disease, normal mRNA and protein are expressed in patient cells. To date, the diagnosis of FD has been limited to individuals of Ashkenazi Jewish descent and identification of the gene has led to widespread diagnostic and carrier testing in this population. In this report, we describe the first non-Jewish IKBKAP mutation, a proline to leucine missense mutation in exon 26, P914L. This mutation is of particular significance because it was identified in a patient who lacks one of the cardinal diagnostic criteria for the disease-pure Ashkenazi Jewish ancestry. In light of this fact, the diagnostic criteria for FD must be expanded. Furthermore, in order to ensure carrier identification in all ethnicities, this mutation must now be considered when screening for FD.     

Tay-Sachs disease in an Arab family due to c.78G>A HEXA nonsense mutation encoding a p.W26X early truncation enzyme peptide

Tay-Sachs disease (TSD), a pan-ethnic, autosomal recessive, neurodegenerative, lysosomal disease, results from deficient β-hexosaminidase A activity due to β-hexosaminidase α-subunit (HEXA) mutations. Prenatal/premarital carrier screening programs in the Ashkenazi Jewish community have markedly reduced disease occurrence. We report the first Jordanian Arab TSD patient diagnosed by deficient β-hexosaminidase A activity. HEXA mutation analysis revealed homozygosity for a nonsense mutation, c.78G>A (p.W26X). Previously reported in Arab patients, this mutation is a candidate for TSD screening in Arab populations.     

Prevalence of known mutations in the familial Mediterranean fever gene (MEFV) in various carrier screening populations

PurposeTo determine the carrier frequency of familial Mediterranean fever (FMF) mutations of individuals in three different US testing populations: Cystic fibrosis, Factor V Leiden, and Ashkenazi Jews.MethodsDNA samples from 1234 anonymous samples were screened for 12 FMF mutations using a laboratory-developed test.ResultsGenotyping revealed carrier frequencies of 1:16, 1:46, and 1:8, respectively.ConclusionMEFV mutation frequency seems to correlate positively with Mediterranean influence of the tested population and the high overall carrier rate for MEFV mutations in the Factor V Leiden testing population (1:46) suggests that the disease may be under-diagnosed in the US population or that the mutant alleles have a low penetrance.     

Comparison of enzyme and DNA analysis in a Tay-Sachs disease carrier screening program.

Tay-Sachs disease (GM2 gangliosidosis, type 1; TSD) is an autosomal recessive GM2 gangliosidosis resulting from the deficient activity of the lysosomal hydrolase beta-hexosaminidase A (Hex A). With a carrier frequency estimated at 1 in 25, it is a common lysosomal disorder in the Ashkenazi Jewish population. Tay-Sachs disease has provided the prototype for the prevention of severe recessive genetic diseases. Molecular analysis of the Hex A gene (HEXA) of Ashkenazi Jewish individuals affected with Tay-Sachs disease revealed that three common mutations cause the infantile and adult onset forms of the disease; a four base insertion in exon 11, a splice junction mutation in intron 12 and a point mutation in exon 7 (G269S). A study was undertaken to determine whether mutation analysis would be useful in TSD screening programs in identifying carriers and clarifying the status of individuals whose enzyme assays are inconclusive. Ashkenazi Jewish individuals who had been diagnosed as carriers, inconclusives by enzyme assay and non-carriers with low normal enzyme levels in the Mount Sinai Tay-Sachs Disease Prevention Program were examined for the presence of the three mutations using polymerase chain reaction (PCR) and allele specific oligonucleotide (ASO) hybridization. The insertion mutation was present in 29 of 34 carriers and 2 of 36 inconclusive individuals, the splice junction mutation was found in 4 of 34 carriers and the G269S mutation was found in 1 of 34 carriers. Of the 313 non-carrier individuals with normal enzyme activity in the lower normal range, one was positive for the splice junction mutation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Methylenetetrahydrofolate reductase (MTHFR): The incidence of mutations C677T and A1298C in the Ashkenazi Jewish population

The polymorphic mutation C677T in the gene of MTHFR is considered a risk mutation for spina bifida and vascular disease. Another common mutation on the MTHFR gene, A1298C, has also been described as another risk mutation. We studied the frequencies of these two mutations on DNA samples from healthy Jewish individuals and compared them to the frequency of these mutations in DNA samples obtained from healthy individuals in South Texas. The presence of the C677T allele was determined by PCR and Hinf I digestion, and mutation A1298C by PCR and Mbo II digestion. A total of 310 alleles was examined for C677T in the Ashkenazi samples and 400 alleles in the non-Jewish samples. The rate of C677T among the Ashkenazi Jewish alleles was 47.7% as compared to 28.7% among the alleles from the non-Jewish population. The difference is statistically significant, P < 0.0005. Mutation A1298C was examined in 298 alleles of Jewish individuals and 374 alleles of non-Jewish counterparts from Texas. The rate of the A1298C mutation in the Jewish samples was 27.2% whereas in the non-Jewish was 35%. This was also statistically significant, P < 0.031. No individuals were homozygous for both mutations or were found to be homozygous for one mutation with heterozygosity of the other mutation, and that the C677T and the A1298C alleles did not occur in cis position. This study shows a unique distribution of C677T and the A1298C alleles among the Ashkenazi Jews. In spite of high frequency of C677T mutation, spina bifida is less common among Ashkenazi Jews. Further studies are needed to establish whether the C677T and the A1298C mutations have an impact on vascular disease in the Ashkenazi Jewish population. Am. J. Med. Genet. 86:380–384, 1999. © 1999 Wiley-Liss, Inc.     

Monoallelic ABCC8 mutations are a common cause of diazoxide‐unresponsive diffuse form of congenital hyperinsulinism

ABCC8 encodes a subunit of the β‐cell potassium channel (K_ATP) whose loss of function is responsible for congenital hyperinsulinism (CHI). Patients with two recessive mutations of ABCC8 typically have severe diffuse forms of CHI unresponsive to diazoxide. Some dominant ABCC8 mutations are responsible for a subset of diffuse diazoxide‐unresponsive forms of CHI. We report the analysis of 21 different ABCC8 mutations identified in 25 probands with diazoxide‐unresponsive diffuse CHI and carrying a single mutation in ABCC8. Nine missense ABCC8 mutations were subjected to in vitro expression studies testing traffic efficiency and responses of mutant channels to activation by MgADP and diazoxide. Eight of the 9 missense mutations exhibited normal trafficking. Seven of the 8 mutants reaching the plasma membrane had dramatically reduced response to MgADP or to diazoxide (<10% of wild‐type response). In our cohort, dominant K_ATP mutations account for 22% of the children with diffuse unresponsive‐diazoxide CHI. Their clinical phenotype being indistinguishable from that of children with focal CHI and diffuse CHI forms due to two recessive K_ATP mutations, we show that functional testing is essential to make the most reliable diagnosis and offer appropriate genetic counseling.     

Methylenetetrahydrofolate reductase (MTHFR): the incidence of mutations C677T and A1298C in the Ashkenazi Jewish population.

The polymorphic mutation C677T in the gene of MTHFR is considered a risk mutation for spina bifida and vascular disease. Another common mutation on the MTHFR gene, A1298C, has also been described as another risk mutation. We studied the frequencies of these two mutations on DNA samples from healthy Jewish individuals and compared them to the frequency of these mutations in DNA samples obtained from healthy individuals in South Texas. The presence of the C677T allele was determined by PCR and Hinf I digestion, and mutation A1298C by PCR and Mbo II digestion. A total of 310 alleles was examined for C677T in the Ashkenazi samples and 400 alleles in the non-Jewish samples. The rate of C677T among the Ashkenazi Jewish alleles was 47.7% as compared to 28.7% among the alleles from the non-Jewish population. The difference is statistically significant, P < 0.0005. Mutation A1298C was examined in 298 alleles of Jewish individuals and 374 alleles of non-Jewish counterparts from Texas. The rate of the A1298C mutation in the Jewish samples was 27.2% whereas in the non-Jewish was 35%. This was also statistically significant, P < 0.031. No individuals were homozygous for both mutations or were found to be homozygous for one mutation with heterozygosity of the other mutation, and that the C677T and the A1298C alleles did not occur in cis position. This study shows a unique distribution of C677T and the A1298C alleles among the Ashkenazi Jews. In spite of high frequency of C677T mutation, spina bifida is less common among Ashkenazi Jews. Further studies are needed to establish whether the C677T and the A1298C mutations have an impact on vascular disease in the Ashkenazi Jewish population.     

Comprehensive population screening in the Ashkenazi Jewish population for recurrent disease‐causing variants

The Ashkenazi Jewish (AJ) population has an increased risk for a variety of recessive diseases due to historical founder effects and genetic drift. For some, the disease‐causing founder mutations have been identified and well‐characterized, but for others, further study is necessary. The purpose of this study is to assess the carrier frequencies of 85 pathogenic variants causative of 29 recessive conditions in the AJ population. Up to 3000 AJ individuals were genotyped by Luminex MagPlex^®‐TAG^? bead array or Agena Bioscience^? MassARRAY assays. We identified seven conditions with carrier frequencies higher than 1 in 100, nine between 1 in 100 and 1 in 200, and four between 1 in 200 and 1 in 500. Variants in nine conditions had a detected carrier rate of less than 1 in 500 or were not identified in approximately 2000 AJ individuals. We assessed the combined AJ carrier frequency for 18 relatively prevalent diseases to be 1 in 6, and the risk of AJ individuals to be a carrier couple for one of these 18 diseases as 1 in 441. We note additional recessive genetic conditions should be considered for AJ carrier screening panels.     

Clinical and molecular characterization of neonatal diabetes and monogenic syndromic diabetes in Asian Indian children

Mutations in the pancreatic ATP sensitive K⁺ channel proteins [sulfonyluea receptor 1 (SUR1) and inward rectifier K⁺ channel Kir6.2 (Kir6.2), encoded by ATP‐binding cassette transporter subfamily C member 8 (ABCC8) and potassium channel J11 (KCNJ11), respectively], are the most common cause of neonatal diabetes. We describe the clinical presentation and molecular characterization of Asian Indian children with neonatal diabetes mellitus and monogenic syndromes of diabetes. We sequenced KCNJ11, ABCC8 and insulin (INS) genes in 33 unrelated Indian probands with onset of diabetes below one year of age. A total of 12 mutations were identified which included ABCC8 mutations in seven, KCNJ11 mutations in three and INS mutations in two children. The Asp212Tyr mutation in ABCC8 was novel. We also detected two novel mutations (Val67Met and Leu19Arg) in children with syndromic forms of diabetes like Berardinelli Seip syndrome [1‐acyl‐sn‐glycerol‐3‐phosphate acyltransferase beta (AGPAT2)] and Fanconi Bickel syndrome [solute carrier family 2A2 (SLC2A2)]. Children carrying the KCNJ11 (Cys42Arg, Arg201Cys) and ABCC8 (Val86Ala, Asp212Tyr) mutations have been successfully switched over from insulin therapy to oral sulfonylurea. Our study is the first large genetic screening study of neonatal diabetes in India.     

Estimating yields of prenatal carrier screening and implications for design of expanded carrier screening panels

PurposePrenatal genetic carrier screening can identify parents at risk of having a child affected by a recessive condition. However, the conditions/genes most appropriate for screening remain a matter of debate. Estimates of carrier rates across genes are needed to guide construction of carrier screening panels.MethodWe leveraged an exome sequencing database (n = 123,136) to estimate carrier rates across six major ancestries for 415 genes associated with severe recessive conditions.ResultsWe found that 32.6% (East Asian) to 62.9% (Ashkenazi Jewish) of individuals are variant carriers in at least one of the 415 genes. For couples, screening all 415 genes would identify 0.17–2.52% of couples as being at risk for having a child affected by one of these conditions. Screening just the 40 genes with carrier rate >1.0% would identify more than 76% of these at-risk couples. An ancestry-specific panel designed to capture genes with carrier rates >1.0% would include 5 to 28 genes, while a comparable panethnic panel would include 40 genes.ConclusionOur work guides the design of carrier screening panels and provides data to assist in counseling prospective parents. Our results highlight a high cumulative carrier rate across genes, underscoring the need for careful selection of genes for screening.     

Ashkenazi Jewish genomic variants: integrating data from the Israeli National Genetic Database and gnomAD

PurposeThe aim of the study was to compare the data for mutations related to clinical disorders reported among Ashkenazi Jewish patients in the Israeli National Genetic Database (INGD) with variants included in the Genome Aggregation Database (gnomAD).MethodsWe extracted data for mutations claimed to cause disorders reported among Ashkenazi Jews from the INGD and searched gnomAD for each of them. We compared the allele frequency of each variant in Ashkenazi Jews with that of other delineated populations.ResultsOf the 58 INGD-reported mutations related to autosomal-dominant disorders, 19 were present in gnomAD (32.8%). Of the 309 mutations related to autosomal-recessive disorders, 240 (77.7%) were variants found in gnomAD. Of these variants, 202 (84.2%) were documented among one or more Ashkenazi individuals. At this point in the INGD, there are 168 Ashkenazi assumed founder mutations in 128 different genes corresponding to 111 autosomal-recessive disorders.ConclusionIntegration of information on mutations among Ashkenazi Jews extracted from the INGD with their population frequency recorded in gnomAD is important for effective straightforward molecular diagnosis as well as for targeted carrier screening either for reproductive decision-making or for implementation of disease-modifying behavior.