Loss of DIAPH1 causes SCBMS, combined immunodeficiency, and mitochondrial dysfunction.

BACKGROUND: Homozygous loss of DIAPH1 results in seizures, cortical blindness, and microcephaly syndrome (SCBMS). We studied 5 Finnish and 2 Omani patients with loss of DIAPH1 presenting with SCBMS, mitochondrial dysfunction, and immunodeficiency. OBJECTIVE: We sought to further characterize phenotypes and disease mechanisms associated with loss of DIAPH1. METHODS: Exome sequencing, genotyping and haplotype analysis, B- and T-cell phenotyping, in vitro lymphocyte stimulation assays, analyses of mitochondrial function, immunofluorescence staining for cytoskeletal proteins and mitochondria, and CRISPR-Cas9 DIAPH1 knockout in heathy donor PBMCs were used. RESULTS: Genetic analyses found all Finnish patients homozygous for a rare DIAPH1 splice-variant (NM_005219:c.684+1G>A) enriched in the Finnish population, and Omani patients homozygous for a previously described pathogenic DIAPH1 frameshift-variant (NM_005219:c.2769delT;p.F923fs). In addition to microcephaly, epilepsy, and cortical blindness characteristic to SCBMS, the patients presented with infection susceptibility due to defective lymphocyte maturation and 3 patients developed B-cell lymphoma. Patients' immunophenotype was characterized by poor lymphocyte activation and proliferation, defective B-cell maturation, and lack of naive T cells. CRISPR-Cas9 knockout of DIAPH1 in PBMCs from healthy donors replicated the T-cell activation defect. Patient-derived peripheral blood T cells exhibited impaired adhesion and inefficient microtubule-organizing center repositioning to the immunologic synapse. The clinical symptoms and laboratory tests also suggested mitochondrial dysfunction. Experiments with immortalized, patient-derived fibroblasts indicated that DIAPH1 affects the amount of complex IV of the mitochondrial respiratory chain. CONCLUSIONS: Our data demonstrate that individuals with SCBMS can have combined immune deficiency and implicate defective cytoskeletal organization and mitochondrial dysfunction in SCBMS pathogenesis.

Screening of hydrocephalus in infants using either WHO or population-based head circumference reference charts.

AIM: The aim was to compare the performances of the World Health Organization (WHO) and population-based (PB) references in the screening for hydrocephalus in infants aged <2 years. METHODS: We collected 341 longitudinal head circumference (HC) measurements of hydrocephalic infants and 120 181 measurements of 15 145 healthy infants from primary care. The measurements were converted into z-scores, and a new screening parameter, change in HC standard deviation score (SDS) over time (ΔHC SDS), was calculated. Comparisons were made using receiver operating characteristics analysis and linear mixed models. RESULTS: The mean HC SDSWHO was 3.5 and the mean HC SDSPB was 2.9 in the hydrocephalic infants, and in healthy children, those numbers were 1.0 SDSWHO and 0 SDSPB , respectively. The best screening accuracy was obtained with the PB reference in combination with the ΔHC SDS parameter (AUC 0.89). The accuracy of the WHO standard could be improved to a similar level by customising the screening cut-offs of HC SDS according to the population and combining screening parameters. CONCLUSIONS: Auxology alone was not sufficient for the screening of hydrocephalus. The WHO standard should be validated in the population, and population-specific cut-offs for normality defined before its introduction.

Transcription factor 7-like 1 is involved in hypothalamo-pituitary axis development in mice and humans.

Aberrant embryonic development of the hypothalamus and/or pituitary gland in humans results in congenital hypopituitarism (CH). Transcription factor 7-like 1 (TCF7L1), an important regulator of the WNT/ß-catenin signaling pathway, is expressed in the developing forebrain and pituitary gland, but its role during hypothalamo-pituitary (HP) axis formation or involvement in human CH remains elusive. Using a conditional genetic approach in the mouse, we first demonstrate that TCF7L1 is required in the prospective hypothalamus to maintain normal expression of the hypothalamic signals involved in the induction and subsequent expansion of Rathke's pouch progenitors. Next, we reveal that the function of TCF7L1 during HP axis development depends exclusively on the repressing activity of TCF7L1 and does not require its interaction with ß-catenin. Finally, we report the identification of two independent missense variants in human TCF7L1, p.R92P and p.R400Q, in a cohort of patients with forebrain and/or pituitary defects. We demonstrate that these variants exhibit reduced repressing activity in vitro and in vivo relative to wild-type TCF7L1. Together, our data provide support for a conserved molecular function of TCF7L1 as a transcriptional repressor during HP axis development in mammals and identify variants in this transcription factor that are likely to contribute to the etiology of CH.

Atypical phenotypes in titinopathies explained by second titin mutations.

OBJECTIVE: Several patients with previously reported titin gene (TTN) mutations causing tibial muscular dystrophy (TMD) have more complex, severe, or unusual phenotypes. This study aimed to clarify the molecular cause of the variant phenotypes in 8 patients of 7 European families. METHODS: Clinical, histopathological, and muscle imaging data of patients and family members were reanalyzed. The titin protein was analyzed by Western blotting and TTN gene by reverse transcription polymerase chain reaction (RT-PCR) and Sanger sequencing. RESULTS: Western blotting showed more pronounced C-terminal titin abnormality than expected for heterozygous probands, suggesting the existence of additional TTN mutations. RT-PCR indicated unequal mRNA expression of the TTN alleles in biopsies of 6 patients, 3 with an limb-girdle muscular dystrophy type 2J (LGMD2J) phenotype. Novel frameshift mutations were identified in 5 patients. A novel A-band titin mutation, c.92167C>T (p.P30723S), was found in 1 patient, and 1 Portuguese patient with a severe TMD phenotype proved to be homozygous for the previously reported Iberian TMD mutation. INTERPRETATION: The unequal expression levels of TTN transcripts in 5 probands suggested severely reduced expression of the frameshift mutated allele, probably through nonsense-mediated decay, explaining the more severe phenotypes. The Iberian TMD mutation may cause a more severe TMD rather than LGMD2J when homozygous. The Finnish patient compound heterozygous for the FINmaj TMD mutation and the novel A-band titin missense mutation showed a phenotype completely different from previously described titinopathies. Our results further expand the complexity of muscular dystrophies caused by TTN mutations and suggest that the coexistence of second mutations may constitute a more common general mechanism explaining phenotype variability.

Analysis of neurodevelopmental outcomes of preadolescents born with extremely low weight revealed impairments in multiple developmental domains despite absence of cognitive impairment.

BACKGROUND AND AIMS: Children with extremely low-birth weight (ELBW) have a high risk for cognitive, motor, and attention impairments and learning disabilities. Longitudinal follow-up studies to a later age are needed in order to increase understanding of the changes in neurodevelopmental trajectories in targeting timely intervention. The aims of this study were to investigate cognitive and motor outcomes, attention-deficit hyperactivity (ADHD) behaviour, school performance, and overall outcomes in a national cohort of ELBW children at preadolescence, and minor neuromotor impairments in a subpopulation of these children and to compare the results with those of full-term controls. The additional aim was to report the overall outcome in all ELBW infants born at 22 to 26 gestational weeks. METHODS: This longitudinal prospective national cohort study included all surviving ELBW (birth weight <1000 g) children born in Finland in 1996 to 1997. No children were excluded from the study. Perinatal, neonatal, and follow-up data up to the age of 5 years of these children were registered in the national birth register. According to birth register, the study population included all infants born at the age under 27 gestational weeks. At 11 years of age general cognitive ability was tested with the Wechsler Intelligence Scale for Children, ADHD behavior evaluated with a report from each child's own teacher (ADHD Rating Scale IV), and school performance with a parental questionnaire. An ELBW subpopulation consisting of a cohort representative children from the two university hospitals from two regions (n = 63) and the age-matched full-term born controls born in Helsinki university hospital (n = 30) underwent Movement Assessment Battery for Children and Touwen neurological examination comprising developmental coordination disorder (DCD) and minor neurological dysfunction (MND), respectively. RESULTS: Of 206 ELBW survivors 122 (73% of eligible) children and 30 (100%) full-term control children participated in assessments. ELBW children had lower full-scale intellectual quotient than controls (t-test, 90 vs 112, P < .001), elevated teacher- reported inattention scores (median = 4.0 vs 1.0, P = .021, r = .20) and needed more educational support (47% vs 17%, OR 4.5, 95% CI 1.6-12.4, P = .02). In the subpopulation, the incidences of DCD were 30% in ELBW and 7% in control children (P = .012, OR 6.0 CI 1.3-27.9), and complex MND 12.5% and 0%, (P = .052; RR 1.1 95% CI 1.04-1.25), respectively. Of survivors born in 24 to 26 gestational weeks, 29% had normal outcome. CONCLUSION: As the majority of the extremely preterm born children had some problems, long-term follow-up is warranted to identify those with special needs and to design individual multidisciplinary support programs.

A novel mutation in the matrix metallopeptidase 2 coding gene associated with intrafamilial variability of multicentric osteolysis, nodulosis, and arthropathy.

BACKGROUND: MONA, which stands for a spectrum of Multicentric Osteolysis, subcutaneous Nodulosis, and Athropathia, is an ultra rare autosomal recessive disorder caused by mutations in the matrix metallopeptidase 2 (MMP2) gene. To date only 44 individuals, carrying 22 different mutations have been reported. Here we report on two brothers with identical homozygous MMP2 gene mutations, but with clearly different phenotypes. METHODS: Genomic DNA was isolated from the affected brothers and the parents. An iliac crest bone biopsy was taken from the younger patient (index case). The level of matrix metallopeptidase 2 enzyme (MMP2) in serum and synovial fluid of the younger patient was analyzed using gelatin zymography. RESULTS: The DNA analysis revealed a homozygous c.1188C>A transversion on exon 8 of the gene. The affected brothers had the same homozygous variant and the parents were heterozygous to this variant. This variant has been reported as a compound heterozygous mutation on one individual resulting in scleroderma like skin thickening. Bone histomorphometry indicated increased trabecular bone remodeling and turnover. The zymography revealed that the level of MMP2 was completely nonmeasurable in the serum and only a minor gelatinolytic protein band of about similar molecular weight as MMP2 was found in the synovial fluid. CONCLUSIONS: Both the age at the onset and the phenotypic severity of the syndrome in these two brothers were different despite identical genotypes. The younger patients had corneal opacities leading to deteriorating visual acuity. For the first time in this disease, opacities were successfully treated with corneal transplantations.

Evidence of a founder effect for the RETGC1 (GUCY2D) 2943DelG mutation in Leber congenital amaurosis pedigrees of Finnish origin.

Leber congenital amaurosis (LCA) is the earliest and most severe form of all inherited retinal dystrophies. It is a genetically heterogeneous condition as six disease-causing genes have been hitherto identified. Among them, RETGC1 (GUCY2D), is more frequently implicated in our series of LCA patients. Interestingly, 70 % of the families with RETGC1 mutations are originating from Mediterranean countries, the remaining families (30%) being originating from various countries across the world. Here, we report, the identification of the same homozygous RETGC1 nonsense mutation in three unrelated and non-consanguineous LCA families of Finnish origin, suggesting a founder effect. Interestingly, no linkage desequilibrium was found using polymorphic markers flanking the RETGC1 gene, supporting the view that the mutation is very ancient. Haplotype studies and Bayesian calculation point the founder mutation to 150 generations (95% credible interval 80-240 generations), i.e., 3000 years ago.

Leber congenital amaurosis: comprehensive survey of the genetic heterogeneity, refinement of the clinical definition, and genotype-phenotype correlations as a strategy for molecular diagnosis.

Leber congenital amaurosis (LCA) is the earliest and most severe form of all inherited retinal dystrophies, responsible for congenital blindness. Disease-associated mutations have been hitherto reported in seven genes. These genes are all expressed preferentially in the photoreceptor cells or the retinal pigment epithelium but they are involved in strikingly different physiologic pathways resulting in an unforeseeable physiopathologic variety. This wide genetic and physiologic heterogeneity that could largely increase in the coming years, hinders the molecular diagnosis in LCA patients. The genotyping is, however, required to establish genetically defined subgroups of patients ready for therapy. Here, we report a comprehensive mutational analysis of the all known genes in 179 unrelated LCA patients, including 52 familial and 127 sporadic (27/127 consanguineous) cases. Mutations were identified in 47.5% patients. GUCY2D appeared to account for most LCA cases of our series (21.2%), followed by CRB1 (10%), RPE65 (6.1%), RPGRIP1 (4.5%), AIPL1 (3.4%), TULP1 (1.7%), and CRX (0.6%). The clinical history of all patients with mutations was carefully revisited to search for phenotype variations. Sound genotype-phenotype correlations were found that allowed us to divide patients into two main groups. The first one includes patients whose symptoms fit the traditional definition of LCA, i.e., congenital or very early cone-rod dystrophy, while the second group gathers patients affected with severe yet progressive rod-cone dystrophy. Besides, objective ophthalmologic data allowed us to subdivide each group into two subtypes. Based on these findings, we have drawn decisional flowcharts directing the molecular analysis of LCA genes in a given case. These flowcharts will hopefully lighten the heavy task of genotyping new patients but only if one has access to the most precise clinical history since birth.

Connexin 26 mutations and nonsyndromic hearing impairment in northern Finland.

OBJECTIVE: The aims of the present study were to evaluate the role of the gap junction protein beta-2 gene (GJB2), encoding connexin 26 (Cx26), in children with moderate to profound prelingual nonsyndromic sensorineural hearing impairment (HI) and to investigate the carrier frequencies of the GJB2 gene mutations in a control population in Northern Finland. METHODS: Mutation analysis was performed by direct sequencing and carrier detection by conformation sensitive gel electrophoresis further confirmed by direct sequencing. RESULTS: Cx26 mutations were found in 15 of 71 (21.1%) (67 families) children with HI. Homozygosity for the mutation 35delG was shown to be the cause of HI in 13 of 15 (86.7%) children. Homozygosity for the M34T genotype was found in one child, and compound heterozygosity for the M34T/V37I genotype was found in another. Five families of those with suspected familial HI (29.4%) and six families out of those with sporadic HI (12.0%) had a homozygous or compound heterozygous mutation. The carrier frequency for the mutation 35delG was 1 of 78 (4 of 313) and that for the M34T was 1 of 26 (12 of 313). CONCLUSION: 35delG/35delG genotype was found to be a significant cause of moderate to profound prelingual nonsyndromic sensorineural HI in Northern Finland. M34T/M34T genotype was seen in only one child, but the carrier frequency of the M34T allele was about three times higher than that of the 35delG mutation.

Homozygous M34T mutation of the GJB2 gene associates with an autosomal recessive nonsyndromic sensorineural hearing impairment in Finnish families.

CONCLUSION: The genetic and audiological data support the hypothesis that the p.M34T is a pathogenic mutation in the Finnish population. The p.M34T mutation displays an autosomal recessive pattern of inheritance and is associated with mild to moderate nonsyndromic sensorineural hearing impairment (SNHI) in the homozygous state. The audiograms often display a hearing impairment notch at 2-4 kHz in young patients, which may aid in the early diagnosis. OBJECTIVES: The aim of the study was to assess whether the p.M34T mutation in the GJB2 gene may associate with nonsyndromic SNHI. METHODS: We systematically reviewed the families with children diagnosed with nonsyndromic SNHI caused by a homozygous p.M34T mutation at the Kuopio and Oulu University Hospital Clinics. The children were re-examined and audiological and genetic data were obtained from their parents and healthy siblings to study genotype-phenotype correlation. RESULTS: We describe 11 patients from 6 families including 5 sibling pairs from 6 to 23 years of age with homozygous p.M34T genotype all having mild nonsyndromic SNHI. In addition, we found three patients with compound p.M34T mutation also exhibiting mild to moderate SNHI.

Prevalence and etiology of congenital or early acquired hearing impairment in Eastern Finland.

OBJECTIVE: The purpose of this study was to determine the prevalence and etiology of congenital or early acquired bilateral sensorineural hearing impairment (SNHI) in children born from 1988 to 2002 in the district of Kuopio University Hospital, Finland, and to compare the results with those from an earlier 14-year period in the same region and similar population. METHODS: The data were collected retrospectively from Hospital Records. The degree of hearing impairment was based on average air conduction threshold calculated over the frequencies 0.5, 1, 2 and 4 kHz in the better hearing ear. Hearing impairment was classified as mild (≥ 20-39 dB), moderate (40-69 dB), severe (70-95 dB) and profound (>95 dB). RESULTS: We identified 92 children with bilateral SNHI diagnosed before the age of 7 years. The overall prevalence and the prevalence for at least moderate SNHI was 2.1 per 1000 live births and 1.2 per 1000 live births, respectively. We found no differences in the prevalence of SNHI during both study periods. Etiology was genetic in 46%, acquired in 14% and unknown in 40%. Out of the genetic cases 74% were non-syndromic and 26% were syndromic. In comparison to the previous study there was a decline in the proportion of acquired SNHI and the proportion of genetic and unknown cause had increased. Six children in five families had homozygous 35delG mutation and six children in four families presented with a homozygous M34T mutation. CONCLUSIONS: The prevalence of congenital or early acquired SNHI in the Kuopio University Hospital district area has not changed during a period of 29 years. Despite possibility to test the GBJ2 gene, the proportion of hearing impairment of unknown etiology remained high.

Prevalence, segregation, and phenotype of the mitochondrial DNA 3243A>G mutation in children.

OBJECTIVE: We studied the prevalence, segregation, and phenotype of the mitochondrial DNA 3243A>G mutation in children in a defined population in Northern Ostrobothnia, Finland. METHODS: Children with diagnoses commonly associated with mitochondrial diseases were ascertained. Blood DNA from 522 selected children was analyzed for 3243A>G. Children with the mutation were clinically examined. Information on health history before the age of 18 years was collected from previously identified adult patients with 3243A>G. Mutation segregation analysis in buccal epithelial cells was performed in mothers with 3243A>G and their children whose samples were analyzed anonymously. RESULTS: Eighteen children were found to harbor 3243A>G in a population of 97,609. A minimum estimate for the prevalence of 3243A>G was 18.4 in 100,000 (95% confidence interval, 10.9-29.1/100,000). Information on health in childhood was obtained from 37 adult patients with 3243A>G. The first clinical manifestations appearing in childhood were sensorineural hearing impairment, short stature or delayed maturation, migraine, learning difficulties, and exercise intolerance. Mutation analysis from 13 mothers with 3243A>G and their 41 children gave a segregation rate of 0.80. The mothers with heteroplasmy greater than 50% tended to have offspring with lower or equal heteroplasmy, whereas the opposite was true for mothers with heteroplasmy less than or equal to 50% (p = 0.0016). INTERPRETATION: The prevalence of 3243A>G is relatively high in the pediatric population, but the morbidity in children is relatively low. The random genetic drift model may be inappropriate for the transmission of the 3243A>G mutation.

GJB2 mutations and degree of hearing loss: a multicenter study.

Hearing impairment (HI) affects 1 in 650 newborns, which makes it the most common congenital sensory impairment. Despite extraordinary genetic heterogeneity, mutations in one gene, GJB2, which encodes the connexin 26 protein and is involved in inner ear homeostasis, are found in up to 50% of patients with autosomal recessive nonsyndromic hearing loss. Because of the high frequency of GJB2 mutations, mutation analysis of this gene is widely available as a diagnostic test. In this study, we assessed the association between genotype and degree of hearing loss in persons with HI and biallelic GJB2 mutations. We performed cross-sectional analyses of GJB2 genotype and audiometric data from 1,531 persons, from 16 different countries, with autosomal recessive, mild-to-profound nonsyndromic HI. The median age of all participants was 8 years; 90% of persons were within the age range of 0-26 years. Of the 83 different mutations identified, 47 were classified as nontruncating, and 36 as truncating. A total of 153 different genotypes were found, of which 56 were homozygous truncating (T/T), 30 were homozygous nontruncating (NT/NT), and 67 were compound heterozygous truncating/nontruncating (T/NT). The degree of HI associated with biallelic truncating mutations was significantly more severe than the HI associated with biallelic nontruncating mutations (P<.0001). The HI of 48 different genotypes was less severe than that of 35delG homozygotes. Several common mutations (M34T, V37I, and L90P) were associated with mild-to-moderate HI (median 25-40 dB). Two genotypes--35delG/R143W (median 105 dB) and 35delG/dela(GJB6-D13S1830) (median 108 dB)--had significantly more-severe HI than that of 35delG homozygotes.

Childhood-onset osteoarthritis, tall stature, and sensorineural hearing loss associated with Arg75-Cys mutation in procollagen type II gene (COL2A1).

OBJECTIVE: To define the clinical, radiologic, and molecular genetic characteristics of a family with early progressive osteoarthritis mimicking childhood rheumatoid arthritis, Scheuermann-like changes of the spine, tall stature, short 3 and 4 metatarsals, and moderate sensorineural hearing loss. METHODS: We describe a 22-year-old woman and her 54-year-old mother with early progressive osteoarthritis mimicking childhood rheumatoid arthritis. The index case, her mother, and 3 other family members underwent a physical examination, anthropometric measurements, and radiologic studies. Their DNA was sequenced for the procollagen type II (COL2A1) gene. RESULTS: Mild scoliosis was noticed in the proband at the age of 6 years, and at the age of 7 years large Schmorl's nodes were found in the vertebrae L1-2. At the age of 11 years, changes resembling Scheuermann's disease were seen, mostly in the thoracic vertebrae. At the same age, she began to have arthralgia in the weight-bearing joints and osteoarthritis progressed fast, necessitating a hip prosthesis at the age of 18 years. The proband and her mother had bilateral sensorineural hearing loss of moderate degree. Both mother and daughter had an Arg75-Cys mutation in the COL2A1 gene. CONCLUSION: This family is the fourth example of the Arg75-Cys mutation in the COL2A1 gene, which appears to lead to a clearly recognizable phenotype. The finding suggests that sensorineural hearing loss may be a part of this syndrome.

Growth and biochemical markers of growth in children with snoring and obstructive sleep apnea.

OBJECTIVE: The pathophysiological mechanisms of growth impairment frequently associated with the obstructive sleep apnea syndrome (OSAS) in children are poorly defined. The main objective of this study was to evaluate whether nighttime upper airway obstruction attributable to adenotonsillar hypertrophy and subsequent surgical treatment affect the circulating concentrations of insulin-like growth factor-I (IGF-I) and IGF-binding protein 3 (IGFBP-3) along with other growth parameters in children. PATIENTS AND METHODS: We initially studied 70 children (mean age: 5.8 years; range: 2.4-10.5 years) admitted to a university hospital because of clinical symptoms of OSAS. Their sleep was monitored with a 6-channel computerized polygraph. Data on anthropometry and circulating concentrations of IGF-I and IGFBP-3 were generated and compared with corresponding characteristics in control children (N = 35). Thirty children with an obstructive apnea-hypopnea index (OAHI) of 1 or more were categorized as children with OSAS (mean OAHI: 5.4 [95% confidence interval for mean (CI): 3.8-6.9]), whereas 40 children with an OAHI of <1 were considered as primary snorers (PS) (mean OAHI 0.13 [95% CI: 0.05-0.21]). Nineteen children with OAHI >2 underwent adenotonsillectomy attributable to OSAS and were reassessed 6 months later together with 34 nonoperated children with OAHI <2. RESULTS: There were no initial differences in relative height and weight for height between the 3 groups of children. No differences were observed in peripheral IGF-I concentrations, but both OSAS and PS children had reduced peripheral IGFBP-3 levels. The operated children with initial OSAS experienced a highly significant reduction in their OAHI from 7.1 (95% CI: 5.1-9.1) to 0.37 (95% CI: 0.2-0.95). Weight-for-height, body mass index, body fat mass, and fat-free mass increased during the follow-up in the operated children with OSAS, whereas only fat-free mass and relative height increased in the PS children. Both the IGF-I and the IGFBP-3 concentrations increased significantly in the operated children, whereas no significant changes were seen in the PS children. CONCLUSIONS: These observations indicate that growth hormone secretion is impaired in children with OSAS and PS. Respiratory improvement after adenotonsillectomy in children with OSAS results in weight gain and restored growth hormone secretion.