Identification and Biochemical Characterization of a Novel Mutation in DDX11 Causing Warsaw Breakage Syndrome

Mutations in the gene encoding the iron–sulfur‐containing DNA helicase DDX11 (ChlR1) were recently identified as a cause of a new recessive cohesinopathy, Warsaw breakage syndrome (WABS), in a single patient with severe microcephaly, pre‐ and postnatal growth retardation, and abnormal skin pigmentation. Here, using homozygosity mapping in a Lebanese consanguineous family followed by exome sequencing, we identified a novel homozygous mutation (c.788G>A [p.R263Q]) in DDX11 in three affected siblings with severe intellectual disability and many of the congenital abnormalities reported in the WABS original case. Cultured lymphocytes from the patients showed increased mitomycin C‐induced chromosomal breakage, as found in WABS. Biochemical studies of purified recombinant DDX11 indicated that the p.R263Q mutation impaired DDX11 helicase activity by perturbing its DNA binding and DNA‐dependent ATP hydrolysis. Our findings thus confirm the involvement of DDX11 in WABS, describe its phenotypical spectrum, and provide novel insight into the structural requirement for DDX11 activity.     

Carrier screening of RTEL1 mutations in the Ashkenazi Jewish population

Hoyeraal–Hreidarsson syndrome (HH) is a clinically severe variant of dyskeratosis congenita (DC), characterized by cerebellar hypoplasia, microcephaly, intrauterine growth retardation, and severe immunodeficiency in addition to features of DC. Germline mutations in the RTEL1 gene have recently been identified as causative of HH. In this study, the carrier frequency for five RTEL1 mutations that occurred in individuals of Ashkenazi Jewish descent was investigated in order to advise on including them in existing clinical mutation panels for this population. Our screening showed that the carrier frequency for c.3791G>A (p.R1264H) was higher than expected, 1% in the Ashkenazi Orthodox and 0.45% in the general Ashkenazi Jewish population. Haplotype analyses suggested the presence of a common founder. We recommend that the c.3791G>A RTEL1 mutation be considered for inclusion in carrier screening panels in the Ashkenazi population.     

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.     

A homozygous mutation in SLC1A4 in siblings with severe intellectual disability and microcephaly

We performed exome analysis in two affected siblings with severe intellectual disability (ID), microcephaly and spasticity from an Ashkenazi Jewish consanguineous family. We identified only one rare variant, a missense in SLC1A4 (c. 766G>A [p. E256K]), that is homozygous in both siblings but not in any of their 11 unaffected siblings or their parents (Logarithm of odds, LOD score: 2.6). This variant is predicted damaging. We genotyped 450 controls of Ashkenazi Jewish ancestry and identified only 5 individuals who are heterozygous for this variant (minor allele frequency: 0.0056). SLC1A4 (ASCT1) encodes a transporter for neutral aminoacids such as alanine, serine, cysteine and threonine. l ‐Serine is essential for neuronal survival and differentiation. Indeed, l ‐serine biosynthesis disorders affect brain development and cause severe ID. In the brain, l ‐serine is synthesized in astrocytes but not in neurons. It has been proposed that ASCT1 mediates the uptake of l ‐serine into neurons and the release of glia‐borne l ‐serine to neighboring cells. SLC1A4 disruption may thus impair brain development and function by decreasing the levels of l ‐serine in neurons. The identification of additional families with mutations in SLC1A4 would be necessary to confirm its involvement in ID.     

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.     

Chinese newborn screening for the incidence of G6PD deficiency and variant of G6PD gene from 2013 to 2017

Glucose‐6‐phosphate dehydrogenase (G6PD) deficiency is one of the most common X‐linked enzymopathies caused by G6PD gene variant. We aimed to provide the characteristics of G6PD deficiency and G6PD gene variant distribution in a large Chinese newborn screening population. We investigated the prevalence of G6PD in China from 2013 to 2017. Then, we examined G6PD activity and G6PD gene in representative Chinese birth cohort to explore the distribution of G6PD gene variant in 2016. We then performed multicolor melting curve analysis to classify G6PD gene variants in 10,357 neonates with activity‐confirmed G6PD deficiency, and DNA Sanger sequencing for G6PD coding exons if hot site variants were not found. The screened population, organizations, and provinces of G6PD deficiency were increased from 2013 to 2017 in China. The top five frequency of G6PD gene variants were c.1376G>T, c.1388G>A, c.95A>G, c.1024C>T, and c.871G>A and varied in different provinces, with regional and ethnic features, and four pathogenic variant sites (c.152C>T, c.290A>T, c.697G>C, and c.1285A>G) were first reported. G6PD deficiency mainly occurs in South China, and the frequency of G6PD gene variant varies in different regions and ethnicities.     

Refining the phenotype of the THG1L (p.Val55Ala mutation)‐related mitochondrial autosomal recessive congenital cerebellar ataxia

Roughly 40 genes have been linked to autosomal recessive (AR) ataxia syndromes. Of these, at least 10 encode gene products localizing to the mitochondrion. tRNA‐histidine guanylyltransferase 1 like (THG1L) localizes to the mitochondrion and catalyzes the 3′–5′ addition of guanine to the 5′‐end of tRNA‐histidine. Previously, three siblings with early onset cerebellar dysfunction, developmental delay, pyramidal signs, and cerebellar atrophy on brain magnetic resonance imaging (MRI) were reported to carry homozygous V55A mutations in THG1L. Fibroblasts derived from these individuals showed abnormal mitochondrial networks when subjected to obligatory oxidative phosphorylation. A carrier rate of 0.8%, but no THG1L V55A homozygotes, was found in a cohort of 3,232 unrelated Ashkenazi Jewish individuals, and no homozygotes were found in Exac or gnomAD. This variant is reported with an allelic frequency of 0.02% in Exac, and is not listed in gnomAD. A similar phenotype was recently reported for another, homozygous variant p.L294P was reported with a similar, but more severely affected phenotype [Shaheen et al. (2019); Genetics in Medicine 21: 545–552]. Here, we report two additional Ashkenazi Jewish patients, carrying the same homozygous V55A mutation. We present bioinformatic analyses of the V55A mutation demonstrating high conservation in metazoan species. We refine the clinical and radiological phenotype and discuss the uniqueness of the clinical course of this novel mitochondrial AR ataxia in comparison to the diverse molecular etiologies and clinical phenotypes of other known mitochondrial AR ataxias.     

Expanding the spectrum of CEP55‐associated disease to viable phenotypes

Homozygosity for nonsense variants in CEP55 has been associated with a lethal condition characterized by multinucleated neurons, anhydramnios, renal dysplasia, cerebellar hypoplasia, and hydranencephaly (MARCH syndrome) also known as Meckel‐like syndrome. Missense variants in CEP55 have not previously been reported in association with disease. Here we describe seven living individuals from five families with biallelic CEP55 variants. Four unrelated individuals with microcephaly, speech delays, and bilateral toe syndactyly all have a common CEP55 variant c.70G>A p.(Glu24Lys) in trans with nonsense variants. Three siblings are homozygous for a consensus splice site variant near the end of the gene. These affected girls all have severely delayed development, microcephaly, and varying degrees of lissencephaly/pachygyria. Here we compare our seven patients with three previously reported families with a prenatal lethal phenotype (MARCH syndrome/Meckel‐like syndrome) due to homozygous CEP55 nonsense variants. Our series suggests that individuals with compound heterozygosity for nonsense and missense variants in CEP55 have a different viable phenotype. We show that homozygosity for a splice variant near the end of the CEP55 gene is also compatible with life.     

Carrier Frequency of the Bloom SyndromeblmMutation in the Ashkenazi Jewish Population

Bloom syndrome is more common in individuals of Ashkenazi Jewish descent than in any other population, and one particular mutation in the Bloom syndrome gene,blm^Ash,is homozygous in nearly all Ashkenazi Jewish persons with Bloom syndrome. We have determined the frequency ofblm^Ashin 1491 Ashkenazi Jewish persons with no known history of Bloom syndrome and found that 1 in 107 persons was heterozygous. Although not common, genetic screening for Bloom syndrome is feasible in this population.     

MICU1 mutation: a genetic cause for a type of neuromuscular disease in children

Two unrelated patients, presenting with significant global developmental delay, severe progressive microcephaly, seizures, spasticity and thin corpus callosum (CC) underwent trio whole‐exome sequencing. No candidate variant was found in any known genes related to the phenotype. However, crossing the data of the patients illustrated that they both manifested pathogenic variants in the SLC1A4 gene which codes the ASCT1 transporter of serine and other neutral amino acids. The Ashkenazi patient is homozygous for a deleterious missense c.766G>A, p.(E256K) mutation whereas the Ashkenazi‐Iraqi patient is compound heterozygous for this mutation and a nonsense c.945delTT, p.(Leu315Hisfs*42) mutation. Structural prediction demonstrates truncation of significant portion of the protein by the nonsense mutation and speculates functional disruption by the missense mutation. Both mutations are extremely rare in general population databases, however, the missense mutation was found in heterozygous mode in 1:100 Jewish Ashkenazi controls suggesting a higher carrier rate among Ashkenazi Jews. We conclude that SLC1A4 is the disease causing gene of a novel neurologic disorder manifesting with significant intellectual disability, severe postnatal microcephaly, spasticity and thin CC. The role of SLC1A4 in the serine transport from astrocytes to neurons suggests a possible pathomechanism for this disease and implies a potential therapeutic approach.     

Carrier screening in the Mexican Jewish community using a pan-ethnic expanded carrier screening NGS panel

PurposeThe Mexican Jewish community (MJC) is a previously uncharacterized, genetically isolated group composed of Ashkenazi and Sephardi-Mizrahi Jews who migrated in the early 1900s. We aimed to determine the heterozygote frequency of disease-causing variants in 302 genes in this population.MethodsWe conducted a cross-sectional study of the MJC involving individuals representing Ashkenazi Jews, Sephardi-Mizrahi Jews, or mixed-ancestry Jews. We offered saliva-based preconception pan-ethnic expanded carrier screening, which examined 302 genes. We analyzed heterozygote frequencies of pathogenic/likely pathogenic variants and compared them with those in the Genome Aggregation Database (gnomAD).ResultsWe recruited 208 participants. The carrier screening results showed that 72.1% were heterozygous for at least 1 severe disease-causing variant in 1 of the genes analyzed. The most common genes with severe disease-causing variants were CFTR (16.8% of participants), MEFV (11.5%), WNT10A (6.7%), and GBA (6.7%). The allele frequencies were compared with those in the gnomAD; 85% of variant frequencies were statistically different from those found in gnomAD (P <.05). Finally, 6% of couples were at risk of having a child with a severe disorder.ConclusionThe heterozygote frequency of at least 1 severe disease-causing variant in the MJC was 72.1%. The use of carrier screening in the MJC and other understudied populations could help parents make more informed decisions.     

In Silico Functional Meta‐Analysis of 5,962 ABCA4 Variants in 3,928 Retinal Dystrophy Cases

Variants in the ABCA4 gene are associated with a spectrum of inherited retinal diseases (IRDs), most prominently with autosomal recessive (ar) Stargardt disease (STGD1) and ar cone‐rod dystrophy. The clinical outcome to a large degree depends on the severity of the variants. To provide an accurate prognosis and to select patients for novel treatments, functional significance assessment of nontruncating ABCA4 variants is important. We collected all published ABCA4 variants from 3,928 retinal dystrophy cases in a Leiden Open Variation Database, and compared their frequency in 3,270 Caucasian IRD cases with 33,370 non‐Finnish European control individuals. Next to the presence of 270 protein‐truncating variants, 191 nontruncating variants were significantly enriched in the patient cohort. Furthermore, 30 variants were deemed benign. Assessing the homozygous occurrence of frequent variants in IRD cases based on the allele frequencies in control individuals confirmed the mild nature of the p.[Gly863Ala, Gly863del] variant and identified three additional mild variants (p.(Ala1038Val), c.5714+5G>A, and p.(Arg2030Gln)). The p.(Gly1961Glu) variant was predicted to act as a mild variant in most cases. Based on these data, in silico analyses, and American College of Medical Genetics and Genomics guidelines, we provide pathogenicity classifications on a five‐tier scale from benign to pathogenic for all variants in the ABCA4‐LOVD database.     

RP1L1 Variants are Associated with a Spectrum of Inherited Retinal Diseases Including Retinitis Pigmentosa and Occult Macular Dystrophy

In one consanguineous family with retinitis pigmentosa (RP), a condition characterized by progressive visual loss due to retinal degeneration, homozygosity mapping, and candidate gene sequencing suggested a novel locus. Exome sequencing identified a homozygous frameshifting mutation, c.601delG, p.Lys203Argfs*28, in RP1L1 encoding RP 1‐like1, a photoreceptor‐specific protein. A screen of a further 285 unrelated individuals with autosomal recessive RP identified an additional proband, homozygous for a missense variant, c.1637G>C, p.Ser546Thr, in RP1L1. A distinct retinal disorder, occult macular dystrophy (OCMD) solely affects the central retinal cone photoreceptors and has previously been reported to be associated with variants in the same gene. The association between mutations in RP1L1 and the disorder OCMD was explored by screening a cohort of 28 unrelated individuals with the condition; 10 were found to harbor rare (minor allele frequency ≤0.5% in the 1,000 genomes dataset) heterozygous RP1L1 missense variants. Analysis of family members revealed many unaffected relatives harboring the same variant. Linkage analysis excluded the possibility of a recessive mode of inheritance, and sequencing of RP1, a photoreceptor protein that interacts with RP1L1, excluded a digenic mechanism involving this gene. These findings imply an important and diverse role for RP1L1 in human retinal physiology and disease.     

A founder mutation in COL4A3 causes autosomal recessive Alport syndrome in the Ashkenazi Jewish population

Alport syndrome is an inherited progressive nephropathy arising from mutations in the type IV collagen genes, COL4A3, COL4A4, and COL4A5. Symptoms also include sensorineural hearing loss and ocular lesions. We determined the molecular basis of Alport syndrome in a non‐consanguineous Ashkenazi Jewish family with multiple affected females using linkage analysis and next generation sequencing. We identified a homozygous COL4A3 mutation, c.40_63del, in affected individuals with mutant alleles inherited from each parent on partially conserved haplotypes. Large‐scale population screening of 2017 unrelated Ashkenazi Jewish samples revealed a carrier frequency of 1 in 183 indicating that COL4A3 c.40_63del is a founder mutation which may be a common cause of Alport syndrome in this population. Additionally, we determined that heterozygous mutation carriers in this family do not meet criteria for a diagnosis of Thin Basement Membrane Nephropathy and concluded that carriers of c.40_63del are not likely to develop benign familial hematuria.     

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.     

Explanations for the discrepancy between variant frequency and homozygous disease occurrence: Lessons from Ashkenazi Jewish data

Ample data on recessive disorders among Ashkenazi Jews has been gathered and published through the years. The opportunity to integrate molecular records analyzed in actual affected individuals with data derived from population-documented frequencies enables to compare these figures.We reviewed assumed pathogenic variants reported among patients in the Israeli medical genetic database (IMGD) with a carrier frequency of 1% or more among Ashkenazi Jews in gnomAD. Among the 60 assumed pathogenic variants recorded in IMGD, 15 (25%) had either a disease incidence considerably lower than expected by the calculated carrier frequency (12 variants), or the variant was not characterized in Ashkenazi Jewish patients (three variants).Possible explanations for the rarity or absence of affected individuals despite high carrier frequency include embryonic lethality, clinical variability, and incomplete and age-related penetrance, in addition to the existence of additional assumed pathogenic variants on the founder haplotype, hypomorphic variants or digenic inheritance.The discrepancy in actual versus expected number of patients calls for caution upon designing and choosing targeted genes and recessive mutations for carrier screening.     

PPP1R21 homozygous null variants associated with developmental delay,muscle weakness,distinctive facial features,and brain abnormalities

We present 3 children with homozygous null variants in the PPP1R21 gene. A 3‐year‐old girl had profound developmental delay, hypotonia and weakness, poor feeding, recurrent chest infections and respiratory failure, rotatory nystagmus, absent reflexes, and a homozygous nonsense variant c.2089C>T (p.Arg697*). A 2‐year‐old boy had profound developmental delay, weakness and hypotonia, recurrent chest infections and respiratory distress, undescended testes, rotatory nystagmus, hyporeflexia, and a homozygous nonsense variant c.427C>T (p.Arg143*). An 11‐year‐old girl with profound developmental delay, weakness and hypotonia, stereotypic movements, growth failure, hyporeflexia, and a homozygous frameshift variant c.87_88delAG (p.Gly30Cysfs*4). In addition, these children shared common facial features (thick eyebrows, hypertelorism, broad nasal bridge, short nose with upturned nasal tip and broad low‐hanging columella, thick lips, low‐set ears, and coarse facies with excessive facial hair), and brain abnormalities (cerebellar vermis hypoplasia, ventricular dilatation, and reduced white matter volume). Although PPP1R21 has not yet been linked to human disease, the consistency in the phenotype of individuals from unrelated families, the nature of the variants which result in truncated proteins, and the expected vital role for PPP1R21 in cellular function, all support that PPP1R21 is a novel disease‐associated gene responsible for the phenotype observed in these individuals.     

Incidence and carrier frequency of Sandhoff disease in Saskatchewan determined using a novel substrate with detection by tandem mass spectrometry and molecular genetic analysis

Sandhoff disease is a rare progressive neurodegenerative genetic disorder with a high incidence among certain isolated communities and ethnic groups around the world. Previous reports have shown a high occurrence of Sandhoff disease in northern Saskatchewan. Newborn screening cards from northern Saskatchewan were retrospectively screened in order to investigate the incidence and determine the carrier frequency of Sandhoff disease in these communities. PCR-based screening was conducted for the c.115delG (p.(Val39fs)) variant in the HEXB gene that was previously found in 4 Sandhoff disease patients from this area. The carrier frequency for this allele was estimated to be ~ 1:27. MS/MS-based screening of hexosaminidase activity along with genetic sequencing allowed for the identification of additional variants based on low total hexosaminidase activity and high % hexosaminidase A activity relative to c.115delG carriers. In total 4 pathogenic variants were discovered in the population (c.115delG, c.619A>G, c.1601G>T, and c.1652G>A) of which two are previously unreported (c.1601G>T and c.1652G>A). The combined carrier frequency of these alleles in the study area was estimated at ~ 1:15. Based on the number of cases of Sandhoff disease from this area we estimate the incidence to be ~ 1:390 corresponding to a child being born with the disease every 1–2 years on average. The results from our study were then compared with variants in the HEXB gene from the genomes available from the 1000 Genomes project. A total of 19 HEXB variants were found in the 1092 genomes of which 5 are suspected of having a deleterious effect on hexosaminidase activity. The estimated carrier frequency of Sandhoff disease in Saskatchewan at 1:15 is more than 3 times higher than the carrier frequency in the global sample provided by the 1000 Genomes project at 1:57.     

Pathogenic variants of DNAJC12 and evaluation of the encoded cochaperone as a genetic modifier of hyperphenylalaninemia

Biallelic variants of the gene DNAJC12, which encodes a cochaperone, were recently described in patients with hyperphenylalaninemia (HPA). This paper reports the retrospective genetic analysis of a cohort of unsolved cases of HPA. Biallelic variants of DNAJC12 were identified in 20 patients (generally neurologically asymptomatic) previously diagnosed with phenylalanine hydroxylase (PAH) deficiency (phenylketonuria [PKU]). Further, mutations of DNAJC12 were identified in four carriers of a pathogenic variant of PAH. The genetic spectrum of DNAJC12 in the present patients included four new variants, two intronic changes c.298‐2A>C and c.502+1G>C, presumably affecting the splicing process, and two exonic changes c.309G>T (p.Trp103Cys) and c.524G>A (p.Trp175Ter), classified as variants of unknown clinical significance (VUS). The variant p.Trp175Ter was detected in 83% of the mutant alleles, with 14 cases homozygous, and was present in 0.3% of a Spanish control population. Functional analysis indicated a significant reduction in PAH and its activity, reduced tyrosine hydroxylase stability, but no effect on tryptophan hydroxylase 2 stability, classifying the two VUS as pathogenic variants. Additionally, the effect of the overexpression of DNAJC12 on some destabilizing PAH mutations was examined and a mutation‐specific effect on stabilization was detected suggesting that the proteostasis network could be a genetic modifier of PAH deficiency and a potential target for developing mutation‐specific treatments for PKU.