Aziz Mhanni

SLC39A8 Deficiency: A Disorder of Manganese Transport and Glycosylation

SLC39A8 is a membrane transporter responsible for manganese uptake into the cell. Via whole-exome sequencing, we studied a child that presented with cranial asymmetry, severe infantile spasms with hypsarrhythmia, and dysproportionate dwarfism. Analysis of transferrin glycosylation revealed severe dysglycosylation corresponding to a type II congenital disorder of glycosylation (CDG) and the blood manganese levels were below the detection limit. The variants c.112G>C (p.Gly38Arg) and c.1019T>A (p.Ile340Asn) were identified in SLC39A8. A second individual with the variants c.97G>A (p.Val33Met) and c.1004G>C (p.Ser335Thr) on the paternal allele and c.610G>T (p.Gly204Cys) on the maternal allele was identified among a group of unresolved case subjects with CDG. These data demonstrate that variants in SLC39A8 impair the function of manganese-dependent enzymes, most notably β-1,4-galactosyltransferase, a Golgi enzyme essential for biosynthesis of the carbohydrate part of glycoproteins. Impaired galactosylation leads to a severe disorder with deformed skull, severe seizures, short limbs, profound psychomotor retardation, and hearing loss. Oral galactose supplementation is a treatment option and results in complete normalization of glycosylation. SLC39A8 deficiency links a trace element deficiency with inherited glycosylation disorders.

Autosomal-Recessive Intellectual Disability with Cerebellar Atrophy Syndrome Caused by Mutation of the Manganese and Zinc Transporter Gene SLC39A8

Manganese (Mn) and zinc (Zn) are essential divalent cations used by cells as protein cofactors; various human studies and animal models have demonstrated the importance of Mn and Zn for development. Here we describe an autosomal-recessive disorder in six individuals from the Hutterite community and in an unrelated Egyptian sibpair; the disorder is characterized by intellectual disability, developmental delay, hypotonia, strabismus, cerebellar atrophy, and variable short stature. Exome sequencing in one affected Hutterite individual and the Egyptian family identified the same homozygous variant, c.112G>C (p.Gly38Arg), affecting a conserved residue of SLC39A8. The affected Hutterite and Egyptian individuals did not share an extended common haplotype, suggesting that the mutation arose independently. SLC39A8 is a member of the solute carrier gene family known to import Mn, Zn, and other divalent cations across the plasma membrane. Evaluation of these two metal ions in the affected individuals revealed variably low levels of Mn and Zn in blood and elevated levels in urine, indicating renal wasting. Our findings identify a human Mn and Zn transporter deficiency syndrome linked to SLC39A8, providing insight into the roles of Mn and Zn homeostasis in human health and development.

Cryptic chromosome rearrangements detected by subtelomere assay in patients with mental retardation and dysmorphic features

The regions near telomeres of human chromosomes are gene rich. Chromosome subtelomere rearrangements occur with a frequency of 7–10% in children with mild‐to‐moderate mental retardation (MR) and approximately 50% of cases are familial. Clinical investigation of subtelomere rearrangements is now prompted by fluorescence in situ hybridization (FISH) analysis using specific DNA probes from all relevant chromosome ends. In our study, 40 children were selected for subtelomere assay using either the Chromophore Multiprobe‐T Cytocell device or the VYSIS TelVision probes. Inclusion criteria were: developmental delay or MR; a normal 550 G‐band karyotype; FRAXA negative; and at least one other clinical criterion. Exclusion criteria included an identified genetic or environmental diagnosis. Of the 40 patients analysed, four (10%) were found to have subtelomere rearrangements. Three of 40 (7.5%) were found to have an unbalanced subtelomere rearrangement and one of 40 (2.5%) was found to have an apparently normal variant subtelomere deletion. The first of the three with an unbalanced karyotype was the result of a familial translocation, the second was a de novo finding, and the origin of the third could not be determined. The subtelomere FISH assay detected almost twice the frequency of unbalanced karyotypes as those detected by 550 G‐banding in our cytogenetics laboratory (4.7%). In addition, subtelomere screening was eight times more likely than fragile X screening in our DNA laboratory (1%) to detect genetic abnormalities in mentally handicapped individuals. Our findings support the view that screening for subtelomere rearrangements has a greater positive yield than other commonly used genetic investigations and, if cost and resources permit, should be the next diagnostic test of choice in a child with unexplained MR/dysmorphisms and a normal 550 G‐band karyotype.

Understanding the impact of 1q21.1 copy number variant

Background1q21.1 Copy Number Variant (CNV) is associated with a highly variable phenotype ranging from congenital anomalies, learning deficits/intellectual disability (ID), to a normal phenotype. Hence, the clinical significance of this CNV can be difficult to evaluate. Here we described the consequences of the 1q21.1 CNV on genome-wide gene expression and function of selected candidate genes within 1q21.1 using cell lines from clinically well described subjects.Methods and ResultsEight subjects from 3 families were included in the study: six with a 1q21.1 deletion and two with a 1q21.1 duplication. High resolution Affymetrix 2.7M array was used to refine the 1q21.1 CNV breakpoints and exclude the presence of secondary CNVs of pathogenic relevance. Whole genome expression profiling, studied in lymphoblast cell lines (LBCs) from 5 subjects, showed enrichment of genes from 1q21.1 in the top 100 genes ranked based on correlation of expression with 1q21.1 copy number. The function of two top genes from 1q21.1, CHD1L/ALC1 and PRKAB2, was studied in detail in LBCs from a deletion and a duplication carrier. CHD1L/ALC1 is an enzyme with a role in chromatin modification and DNA damage response while PRKAB2 is a member of the AMP kinase complex, which senses and maintains systemic and cellular energy balance. The protein levels for CHD1L/ALC1 and PRKAB2 were changed in concordance with their copy number in both LBCs. A defect in chromatin remodeling was documented based on impaired decatenation (chromatid untangling) checkpoint (DCC) in both LBCs. This defect, reproduced by CHD1L/ALC1 siRNA, identifies a new role of CHD1L/ALC1 in DCC. Both LBCs also showed elevated levels of micronuclei following treatment with a Topoisomerase II inhibitor suggesting increased DNA breaks. AMP kinase function, specifically in the deletion containing LBCs, was attenuated.ConclusionOur studies are unique as they show for the first time that the 1q21.1 CNV not only causes changes in the expression of its key integral genes, associated with changes at the protein level, but also results in changes in their known function, in the case of AMPK, and newly identified function such as DCC activation in the case of CHD1L/ALC1. Our results support the use of patient lymphoblasts for dissecting the functional sequelae of genes integral to CNVs in carrier cell lines, ultimately enhancing understanding of biological processes which may contribute to the clinical phenotype.

Cytogenetic microarrays in Manitoba patients with developmental delay

To the Editor: Cytogenetic microarrays have revolutionized conventional cytogenetics by increasing the resolution of the standard karyotype from 5–10 to ,1 Mb using microarray comparative genomic hybridization (aCGH) (1). Targeted microarrays, which cover specific areas of the genome with known clinical significance, and whole genome arrays, which span the entire genome but detect copy number polymorphisms/variants/changes (CNVs) of unknown clinical significance, allow for the comprehensive analysis of hundreds or thousands of distinct genomic loci for DNA copy number losses or gains. Conventional cytogenetics identifies 5–10% abnormalities depending on ascertainment criteria, and microarrays can identify an additional 10–15% of previously undetected abnormalities. The purpose of our study was to determine how many Manitoba patients had unrecognized chromosome imbalances detectable by microarray. Inclusion criteria were unexplained developmental delay (DD) or mental retardation (MR), a normal 550 G-band karyotype, fragile X (FMR1) negative, and at least one of the following additional criteria: multiple congenital anomalies; prenatal growth retardation; postnatal growth abnormalities including microcephaly, short or tall stature, or macrocephaly; facial or non-facial dysmorphisms; or a family history of any of the above clinical criteria in a child with unexplained DD or MR. Exclusion criteria included an identified genetic or environmental diagnosis. A total of 74 children were selected for targeted bacterial artificial chromosome (BAC) microarray analysis using TOP (technical only program) at Combimatrix Molecular Diagnostics (Irvine, CA). The microarray platform used at this time was the CA 1500, which is a targeted array of 1500 BAC clones representing greater than 125 constitutional disorders. All patient microarray abnormalities were confirmed by fluorescent in situ hybridization (FISH); microarray, FISH and karyotype analysis were performed on parents of patients with microarray abnormalities. A total of 7 of 74 (10%) of patients had clinically significant microarray abnormalities, with phenotypes compatible with the specific genomic imbalance observed. The observed frequency is consistent with previous reports (2, 3). These abnormalities are summarized in Table 1. Two of 74 (3%) had unbalanced 1p36 deletions (one interstitial, patient 1; one terminal, patient 2), 2 (3%) (patients 3 and 4) had other subtelomere rearrangements, and the remaining 3 (4%) (patients 5, 6, and 7) had various interstitial deletions. Thus, approximately 5% had subtelomere rearrangements, in agreement with previous estimates (1, 2). Parental microarray studies did not detect any unbalanced cases. However, FISH analyses of the parents of patient 4 (Table 1) showed the presence of a paternal subtelomere translocation: t(12;18)(qter2,pter1;pter2,qter1). In addition, karyotyping the parents of patient 3 (Table 1) fortuitously revealed the presence of a paternal t(1;3)(p36.3;p13), which would not have been otherwise detected as both parents had normal confirmatory subtelomere FISH and microarray results. We recommend confirmatory FISH in all parents of a child with an abnormal microarray result. Parental aCGH may not always be necessary but may be required to provide supportive evidence for the association between a CNV and the clinical presentation. Karyotyping of parents is still recommended for balanced rearrangements. Our targeted microarray study detected 23 the frequency of unbalanced abnormalities as found by conventional karyotyping in our Cytogenetics Laboratory ( 5.4%) (unpublished data) and 103 the full FMR1 mutation in our Molecular Diagnostic Laboratory ( 1%) (unpublished data). Our findings suggest that screening for microarray

Matchmaking facilitates the diagnosis of an autosomal-recessive mitochondrial disease caused by biallelic mutation of the tRNA isopentenyltransferase (TRIT1) gene

Deleterious variants in the same gene present in two or more families with overlapping clinical features provide convincing evidence of a disease–gene association; this can be a challenge in the study of ultrarare diseases. To facilitate the identification of additional families, several groups have created “matching” platforms. We describe four individuals from three unrelated families “matched” by GeneMatcher and MatchMakerExchange. Individuals had microcephaly, developmental delay, epilepsy, and recessive mutations in TRIT1. A single homozygous mutation in TRIT1 associated with similar features had previously been reported in one family. The identification of these individuals provides additional evidence to support TRIT1 as the disease‐causing gene and interprets the variants as “pathogenic.” TRIT1 functions to modify mitochondrial tRNAs and is necessary for protein translation. We show that dysfunctional TRIT1 results in decreased levels of select mitochondrial proteins. Our findings confirm the TRIT1 disease association and advance the phenotypic and molecular understanding of this disorder.

Successful therapy for protein-losing enteropathy caused by chronic neuronopathic Gaucher disease

Gaucher disease (OMIM #230800) is caused by β-glucosidase deficiency and primarily involves the mononuclear phagocyte system (also called Reticuloendothelial System or Macrophage System). The disease is classified into three main phenotypes based on the presence or absence of neurological manifestations: non-neuronopathic (type 1), acute neuronopathic (type 2) and chronic neuronopathic (type 3). Typical manifestations include hepatosplenomegaly, skeletal deformities, hematological abnormalities, interstitial lung fibrosis and neurodegeneration in neuronopathic cases. Mesenteric lymphadenopathy with resultant protein losing enteropathy (PLE) has only been rarely described. Mesenteric lymphadenopathy may lead to intestinal lymphatic obstruction and secondary lymphangiectasia resulting in chronic diarrhea, abdominal pain and weight loss. Fecal protein loss with secondary hypoalbuminemia can be significant. We report a male with Chronic Neuronopathic Gaucher disease (GD) (homozygous for c.1448T > C (NM_000157.3) GBA mutation) who at 16 years of age developed intractable abdominal pain, diarrhea and weight loss. This was caused by PLE secondary to intestinal lymphangiectasia caused by calcified mesenteric lymphadenopathy despite prior long term enzyme replacement therapy (ERT) and/or substrate reduction therapy (SRT). His older similarly affected sister who had been receiving treatment with ERT and/or SRT remains stable on these treatments with no evidence of mesenteric lymphadenopathy. Medical management with total parenteral nutrition, daily medium chain triglyceride-oil (MCT) supplementation, low dose oral budesonide, continued oral SRT and an increased dose of parenteral ERT has stabilized his condition with resolution of the gastrointestinal symptoms and appropriate weight gain.

Molecular cytogenetic investigation of two patients with Y chromosome rearrangements and intellectual disability

We describe two males with intellectual disability (ID) and facial dysmorphism, both of whom have non‐mosaic Y chromosome rearrangements resulting in deletions of large portions of the Y chromosome. Patient A, with ID, mild dysmorphism, speech delay, Duane anomaly of the eye, hypermetropia and conductive hearing loss, had two structurally rearranged Y chromosomes resulting in both p and q arm deletions in addition to a Yp duplication. Patient B, also with speech and language delay, developmental delay and short stature, had an interstitial deletion of Yq11.21–11.23. Array‐CGH excluded the presence of additional submicroscopic rearrangements at the 1 Mb resolution level. A review of males with Y chromosome rearrangements and ID was performed. Our study provides a more detailed molecular cytogenetic assessment of Y rearrangements in individuals with ID than has been previously possible, and facilitates assessment and comparison of other individuals with a Y chromosome rearrangement.

Increasing incidence of optic nerve hypoplasia/septo-optic dysplasia spectrum: Geographic clustering in Northern Canada

Introduction Owing to the shared embryonic origin, defects in development of optic nerves are often seen in conjunction with defects affecting the surrounding brain and pituitary gland. Optic nerve hypoplasia (ONH) and septo-optic dysplasia (SOD) represent a clinical spectrum associated with visual, pituitary and severe central nervous system structural abnormalities (SODplus). Based on changing clinical patterns, our primary objective was to examine trends in annual incidence of ONH/SOD and geographical clustering in Manitoba. Methods This was a retrospective 1996 to 2015 chart review with extraction of anthropometric measures, radiologic findings, parental characteristics, endocrinopathies and neurologic symptoms from all involved in care. Postal codes were used to assign map co-ordinates and identify relevant census-based deprivation indices. Results Ninety-three children were identified in our catchment area; Poisson regression confirmed a striking 1.11-fold annual increase (95% confidence interval 1.07 to 1.16) or ~800% over two decades. The annual incidence (averaged 2010 to 2014 chart data) reached 53.3 per 100,000, affecting 1 in 1875 live births. Most (~55%) had SODplus. Common presenting features were hypoglycemia, nystagmus, seizures and developmental delay; 40% had hormone deficiencies; 80% had reduced visual acuity, typically bilateral. Many were premature with young, primiparous mothers. Unhealthy maternal lifestyles and severe material deprivation were noted. There was disproportionate clustering in individuals from Northern Manitoba at three times the average provincial rate. Conclusion We noted a dramatic rise in the annual incidence of ONH/SOD, which was strongly associated with poverty and northern communities. The pattern was consistent with environmental or nutritional etiologies. Many children were severely affected with increased morbidity and health care burdens.

Ataxia with oculomotor apraxia type 2 in the Canadian aboriginal population.

To the Editor: Ataxia with oculomotor apraxia type 2 (AOA2) (OMIM 606002) is an autosomal recessive disorder, representing approximately 8% of non-Friedreich autosomal recessive cerebellar ataxias. It is characterized by ataxia onset between 11 and 22 years, and associated with oculomotor apraxia and elevated serum alpha fetoprotein. It was first described in Japanese (1), Pakistani (2) and French Canadian patients (3). We report on affected siblings from a consanguineous family of Ojibwe Canadian Aboriginal descent. The proband presented at age 16 years with insidious and protracted balance difficulties since age 11 years. By 16 years, his symptoms advanced to a noticeably unsteady gait with occasional falls and deterioration in manual dexterity. He was born to healthy parents who are second cousins. His older sister had similar gait abnormality. Reportedly, a paternal first cousin and two maternal first cousins once removed have a similar phenotype. The parents of these individuals are second cousins. These relatives have not presented for a formal evaluation. On examination, his speech was dysarthric and scanning. There was ocular apraxia with initiation of saccades with a head thrust maneuver. He had sustained gaze-evoked nystagmus with lateral gaze. There were no conjunctival telangiectasias. The fine finger and rapid alternating movements were impaired. He displayed symmetric dysmetria on finger-nose-finger and heel-to-shin testing. His gait and stance were broad in base. He was unable to perform tandem walking. The work up included serum alpha fetoprotein (AFP) which was elevated at 20 μg/l (normal 0.0–7.0 μg/l). Brain magnetic resonance imaging revealed cerebellar atrophy affecting the vermis. Chromosome breakage studies did not show evidence of chromosome instability. Mutation analysis of the SETX gene (Prevention Genetics, Marshfield, WI) was performed by polymerase chain reaction amplification of genomic DNA followed by automated sequencing of all the coding exons, as well as 50 bases of flanking non-coding sequences. This revealed a homozygous c.1406A>G (NM_015046.5) mutation predicted to result in the amino acid substitution p.His469Arg. His affected sister was also homozygous for this mutation. Since the early reports of AOA2 in Japanese (1), Pakastani (2) and French Canadian (3) families, it has now been reported in a wide range of populations. This report is the first to document the disease presence in the Canadian Aboriginal population. To date more than 75 mutations have been identified in more than 100 reported patients worldwide. The vast majority of these mutations are distributed across the entire gene without any reported hot spots. Our patients were homozygous for a c.1406A>G, a mutation that has been reported to be causative of AOA2 in a consanguineous family from Southern Italy (4). This substitution was not found in 200 Italian control chromosomes (4) nor did we identify this change in any of the population databases, including dbSNP, 1000Genomes and Exome Aggregation Consortium. In silico analysis using PolyPhen-2, SIFT and MutationTaster predict p.His469Arg change to be ‘probably damaging’, ‘not tolerated’ and ‘disease causing’, respectively (5–7). Based on the recent ACMG guidelines for interpreting sequence variants (8), the c.1406A>G is likely pathogenic. Consanguinity in this family favors the idea of a founder mutation. The presence of the same mutation in the family from Southern Italy is likely a recurrent event and not derived from a single ancestral mutation. This report supports the panethnic presence of AOA2.