Homozygous Mutations in Thyroid Peroxidase (TPO) in Hypothyroidism with Intellectual Disability, Developmental Delay, and Hearing and Ocular Anomalies in Two Families: Severe Manifestation of Untreated TPO-deficiency Poses a Diagnostic Dilemma.

Intellectual disability (ID) involves compromised intellectual, learning and cognitive skills, and behavioral capabilities with reduced psychomotor skills. One of the preventable causes of ID is congenital hypothyroidism (CH), which may be due to biallelic mutations in thyroid peroxidase (TPO). In low- and middle-income countries with no newborn screening programs, CH poses a great risk of ID and long-term morbidity. We report two large Pakistani families with a total of 16 patients afflicted with CH. Detailed clinical and behavioral assessments, SNP-based homozygosity mapping, linkage analysis, and exome sequencing were performed. Initially, affected individuals were referred as suffering ID (in 11 of 16 patients) and developmental delay (in 14). Secondary/associated features were verbal apraxia (in 13), goiter (in 12), short stature (in 11), limb hypotonia (in 14), no pubertal onset (five of 10 of age ≥14 years), high myopia (in eight), muscle cramps (in six), and in some, variable microcephaly and enuresis/encopresis, fits, chronic fatigue, and other behavioral symptoms, which are not characteristics of CH. Molecular genetic analyses led to the discovery of homozygous variants in TPO: novel missense variant c.719A>G (p.Asp240Gly) in family 1 and rare c.2315A>G (p.Tyr772Cys) in family 2. In low-resource countries where neonatal screening programs do not include a CH test, the burden of neurodevelopmental disorders is likely to be increased due to untreated CH. Secondly, in the background of the high prevalence of recessive disorders due to high parental consanguinity, the severe manifestation of TPO-deficiency mimics a wide range of neurological and other presentations posing a diagnostic dilemma.

High-level gonosomal mosaicism for a pathogenic non-coding CNV deletion of the lung-specific FOXF1 enhancer in an unaffected mother of an infant with ACDMPV.

BACKGROUND: Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) results from haploinsufficiency of the mesenchymal transcription factor FOXF1 gene. To date, only one case of an ACDMPV-causative CNV deletion inherited from a very-low level somatic mosaic mother has been reported. METHODS: Clinical, histopathological, and molecular studies, including whole genome sequencing, chromosomal microarray analysis, qPCR, and Sanger sequencing, followed by in vitro fertilization (IVF) with preimplantation genetic testing (PGT) were used to study a family with a deceased neonate with ACDMPV. RESULTS: A pathogenic CNV deletion of the lung-specific FOXF1 enhancer in the proband was found to be inherited from an unaffected mother, 36% mosaic for this deletion in her peripheral blood cells. The qPCR analyses of saliva, buccal cells, urine, nail, and hair samples revealed 19%, 18%, 15%, 19%, and 27% variant allele fraction, respectively, indicating a high recurrence risk. Grandparental studies revealed that the deletion arose on the mother's paternal chromosome 16. PGT studies revealed 44% embryos with the deletion, reflecting high-level germline mosaicism. CONCLUSION: Our data further demonstrate the importance of parental testing in ACDMPV families and reproductive usefulness of IVF with PGT in families with high-level parental gonosomal mosaicism.

Variable expressivity in a four-generation ACDMPV family with a non-coding hypermorphic SNV in trans to the frameshifting FOXF1 variant.

Heterozygous single nucleotide variants (SNVs) or copy-number variant deletions involving FOXF1 or its distant lung-specific enhancer on chromosome 16q24.1 have been identified in 80-90% of patients with Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV), a lethal neonatal lung developmental disorder. We describe a four-generation family with a deceased ACDMPV neonate, her sibling from the electively terminated pregnancy, healthy mother with a history of pulmonary arterial hypertension (PAH), an unaffected aunt, an aunt deceased due to findings consistent with ACDMPV, and a reportedly unaffected grandmother, all with the frameshifting variant c.881_902dup (p.Gly302Profs*46) in FOXF1, and a deceased great-grandmother with a history of PAH. Genome sequencing analyses in the proband's unaffected mother revealed a non-coding putative regulatory SNV rs560517434-A within the lung-specific distant FOXF1 enhancer in trans to the FOXF1 frameshift mutation. Functional testing of this variant using an in vitro luciferase reporter assay showed that it increased FOXF1 promoter activity 10-fold. Our studies further demonstrate that non-coding SNVs in the FOXF1 enhancer region can rescue the lethal ACDMPV phenotype and support the compound inheritance gene dosage model.

Do paternal deletions involving the FOXF1 locus on chromosome 16q24.1 manifest with more severe non-lung anomalies?

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a rare lethal lung developmental disorder in neonates due to heterozygous loss-of-function of the mesenchymal transcription factor gene, FOXF1. Interestingly, unlike ACDMPV-causing point mutations in FOXF1 that can be inherited from the mother or father, causative copy-number variant (CNV) deletions arise de novo and almost exclusively on chromosome 16 inherited from the mother (n = 50 vs. n = 3). Here, we describe a fourth case of a de novo paternal CNV deletion encompassing FOXF1, its neighboring long non-coding RNA gene FENDRR, and their distant lung-specific enhancer, identified in a 21-week-old fetus with tetralogy of Fallot, gastrointestinal and genitourinary abnormalities, a single umbilical artery, and patchy histopathological findings of ACDMPV in autopsy lung. We also review the ACDMPV-causative CNV deletions detected prenatally and propose that the majority of paternal deletions manifest with more severe additional non-lung abnormalities.

Exacerbation of mild lung disorders to lethal pulmonary hypoplasia by a noncoding hypomorphic SNV in a lung-specific enhancer in trans to the frameshifting TBX4 variant.

Variants involving TBX4 are associated with a wide variety of disorders, including pulmonary arterial hypertension, ischiocoxopodopatellar syndrome (ICPPS)/small patella syndrome (SPS), lethal lung developmental disorders (LLDDs) in neonates, heart defects, and prenatally lethal posterior amelia with pelvic and pulmonary hypoplasia syndrome. The objective of our study was to elucidate the wide variable phenotypic expressivity and incomplete penetrance in a three-generation family with a truncating variant in TBX4. In addition to exome and genome sequencing analyses, a candidate noncoding regulatory single nucleotide variant (SNV) within the lung-specific TBX4 enhancer was functionally tested using an in vitro luciferase reporter assay. A heterozygous frameshift variant c.1112dup (p.Pro372Serfs*14) in TBX4 was identified in patients with mild interstitial lung disease (1), bronchiolitis obliterans (1), recurrent pneumothorax (1), ICPPS/SPS (1), LLDD (2), and in unaffected individuals (4). In two deceased neonates with LLDD, we identified a noncoding SNV rs62069651-C located in trans to the mutated TBX4 allele that reduced the TBX4 promoter activity by 63% in the reporter assay. Our findings provide a functional evidence for the recently reported model of complex compound inheritance in which both TBX4 coding and in trans noncoding hypomorphic variants in the lung-specific enhancer of TBX4 contribute to LLDD.

Mutation in protein disulfide isomerase A3 causes neurodevelopmental defects by disturbing endoplasmic reticulum proteostasis.

Recessive gene mutations underlie many developmental disorders and often lead to disabling neurological problems. Here, we report identification of a homozygous c.170G>A (p.Cys57Tyr or C57Y) mutation in the gene coding for protein disulfide isomerase A3 (PDIA3, also known as ERp57), an enzyme that catalyzes formation of disulfide bonds in the endoplasmic reticulum, to be associated with syndromic intellectual disability. Experiments in zebrafish embryos show that PDIA3C57Y expression is pathogenic and causes developmental defects such as axonal disorganization as well as skeletal abnormalities. Expression of PDIA3C57Y in the mouse hippocampus results in impaired synaptic plasticity and memory consolidation. Proteomic and functional analyses reveal that PDIA3C57Y expression leads to dysregulation of cell adhesion and actin cytoskeleton dynamics, associated with altered integrin biogenesis and reduced neuritogenesis. Biochemical studies show that PDIA3C57Y has decreased catalytic activity and forms disulfide-crosslinked aggregates that abnormally interact with chaperones in the endoplasmic reticulum. Thus, rare disease gene variant can provide insight into how perturbations of neuronal proteostasis can affect the function of the nervous system.

Homozygous deletion of MYADML2 in cranial asymmetry, reduced bone maturation, multiple dislocations, lumbar lordosis, and prominent clavicles.

A null mutation in a patient can facilitate phenotype assignment and uncovers the function of that specific gene. We present five sibs of a consanguineous Pakistani family afflicted with a new syndrome with an unusual combination of skeletal anomalies including cranial asymmetry, fused sagittal sutures deviating from the medial axis, mandibular prognathia, maxillary hypoplasia, misaligned and crowded teeth, delayed bone age, multiple dislocations, hypoplastic and malpositioned patellae, humeral intracondylar fissures, scapular dyskinesis, long limbs, lumbar lordosis, protruding chest, prominent clavicles, short 5th digital rays, and ventral transverse digital creases plus features of cutis laxa. We mapped the disease gene locus to a 3.62-Mb region at 17q25.3 and identified a homozygous deletion of maximal 7.3 kb deduced to totally inactivate MYADML2 and downstream gene PYCR1, biallelic variants in which cause autosomal recessive cutis laxa (ARCL). All five affected sibs had the most common features of ARCL but not many of the less common ones. We attributed the anomalies not typical for ARCL to MYADML2 deficit, because no other genetic defect possibly a candidate to underlie the skeletal phenotype was found. MYADML2 is a gene of unknown function, has not been studied, and has not been associated with disease. Our findings present a possible phenotype for MYADML2 deficit that includes impaired bone patterning and maturation, definitely show that the gene is not essential for survival, and provide a start point for future studies on the function of MYADML2 protein. Detection of new patients is needed to confirm and delineate MYADML2-deficiency phenotype.

Novel EDAR mutation in tooth agenesis and variable associated features.

Tooth agenesis (TA) is the developmental absence of one or more permanent teeth. We report on 10 members of a Pakistani family afflicted with TA with variable associated features inherited in autosomal dominant fashion with full penetrance. The malformation is bilateral in the majority of cases, and hallmark feature is the absence of lateral and central incisors and canines whereas first and second premolars are involved less often. Affected individuals also have pronounced variable features associated with TA such as diastema between central incisors, overgrown labial frenum, peg-shaped lower incisors, delayed exfoliation, over-erupted upper incisors and malocclusion but have no other signs of ectodermal dysplasia. Through linkage analysis coupled with exome sequencing, we identified novel nonsense variant EDAR c.1302G>A, p.(Trp434*). The variant is deduced to create a premature termination codon that leads to the deletion of the 15 C-terminal residues. Heterozygous EDAR variants most commonly cause hypohydrotic ectodermal dysplasia, but recently one nonsense and 10 missense variants have been reported in nonsyndromic TA, some with few mild features of hypohydrotic ectodermal dysplasia. The phenotype in the family we present, the largest with EDAR-related TA reported to date, is highly variable and without any signs of ectodermal dysplasia.

Homozygous mutation in CEP19, a gene mutated in morbid obesity, in Bardet-Biedl syndrome with predominant postaxial polydactyly.

BACKGROUND: Bardet-Biedl syndrome (BBS) is a ciliopathy with extensive phenotypic variability and genetic heterogeneity. We aimed to discover the gene mutated in a consanguineous kindred with multiple cases of a BBS phenotype. METHODS: SNP genotype data were used for linkage analysis and exome sequencing to identify mutations. Modelling and in silico analysis were performed to predict mutation severity. RESULTS: Patients had postaxial polydactyly plus variable other clinical features including rod-cone dystrophy, obesity, intellectual disability, renal malformation, developmental delay, dental anomalies, speech disorder and enlarged fatty liver. The 4.57 Mb disease locus harboured homozygous, truncating CEP19 c.194_195insA (p.Tyr65*) mutation. We also found glioma-associated oncogene homolog 1(GLI1) c.820G>C (p.Gly274Arg) in the homozygous state in most patients. In silico modelling strongly suggests that it is damaging. Also, different combinations of four possible modifier alleles in BBS-related genes were detected. Two are known modifier alleles for BBS, splicing variant CCDC28B c.330C>T and missense MKKS/BBS6 p.Ile339Val, and the others are C8ORF37/BBS21 p.Ala178Val and TMEM67/BBS14 modifier p.Asp799Asp. Some patients carry all those five known/possible modifier alleles. Such variants are highly significantly more abundant in our patients than in a control group. CONCLUSION: CEP19 encodes a centrosomal and ciliary protein, as all BBS genes do. Another truncating mutation p.Arg82* has been reported as responsible for morbid obesity in a family; however, in the family we present, not all homozygotes are obese, although some are severely obese. The variant in GLI1, encoding a transcription factor that localises to the primary cilium and nucleus and is a mediator of the sonic hedgehog pathway, possibly exacerbates disease severity when in the homozygous state.

Progressive SCAR14 with unclear speech, developmental delay, tremor, and behavioral problems caused by a homozygous deletion of the SPTBN2 pleckstrin homology domain.

We report on nine members of a consanguineous Pakistani family with primary presentation of intellectual disability, developmental delay, limb and gait ataxia, behavioral and speech problems, and tremor. By linkage mapping and exome sequencing we identified novel homozygous splicing variant c.6375-1G>C in SPTBN2. To date, only two other SPTBN2 mutations with recessive pattern of inheritance causing SCAR14 (spinocerebellar ataxia, autosomal recessive 14) that manifest with developmental ataxia and cognitive impairment, or cerebellar ataxia, mental retardation, and pyramidal signs have been reported. The mutation we identified is predicted to lead to the deletion of just the pleckstrin homology domain; thus, the earlier onset and more progressive nature of the disease in the presented family, as compared to earlier reports, were unexpected. No other mutation that could possibly explain the features that were unusual for SCAR14-arched palate, limb hypotonia, climacophobia, and behavioral problems-was identified. The disease was more severe in males than females. Our findings expand the recessive SPTBN2 mutation phenotype. We also review SPTBN2 mutation phenotypes. The gene encodes beta-III spectrin, which forms tetramers with alpha-II spectrin. The manifestations of this third recessive mutation suggest that for recessive mutations either no mutant protein is synthesized because the transcript is subject to nonsense-mediated decay or the mutant protein does not bind membrane proteins and, thus, does not exert a negative effect in heterozygotes, whereas the dominant mutations causing SCA5 form defective tetramers that compete with the native tetramers in binding membrane proteins, but are unable to anchor them.