Primary Microcephaly with Novel Variant of MCPH1 Gene in Twins: Both Manifesting in Childhood at the Same Time with Hashimoto's Thyroiditis

Abstract

This study is a clinical report on twin females affected by primary microcephaly who displayed at molecular analysis of heterozygous novel MCPH1 variant. The twins at the age of 10 years developed, in coincidental time, a diagnosis of autoimmune juvenile thyroiditis. The main clinical features presented by the twins consisted of primary microcephaly with occipitofrontal circumference measuring −2 or −3 standard deviation, facial dysmorphism, typical nonsyndromic microcephaly, and mild intellectual disability. Molecular analysis of the major genes involved in primary microcephaly was performed and the following result was found in the twins: MCPH1 ; chr8.6357416; c.2180 C > T (rs 199861426), p.Pro727. Leu; heterozygous; missense; variant of uncertain significance (class 3). At the age of 10 years, the twins started to have, in coincidental time, marked asthenia and episodes of emotiveness, and laboratory exams disclosed a high level of antithyroid peroxidase leading to the diagnosis of autoimmune juvenile thyroiditis with normal thyroid function. The novel heterozygous MCPH1 variant found in the twins may be directly or indirectly involved in the onset of the primary microcephaly. The thyroid disorder in the twins and its onset, in a coincidental time, confirmed the effect of genetic predisposition on the pathogenesis of the immune thyroiditis.

Introduction

Microcephaly is a sign of several disorders, manifesting with different clinical expression more frequently with intellectual disability ranging from mild to severe. Microcephaly is defined as a head circumference measurement more than 2 standard deviation (SD) below the mean for age, sex, and ethnicity. ^{1 2} Correct measurement of the occipitofrontal circumference (OFC) in childhood is of great relevance since an erroneous evaluation of a few centimeters in the range of −2 to −3 SD may result in different prognostic evaluation. Of note, ∼2% of the general population may present with an OFC of −2 SD without presenting cognitive impairment. ^{3 4} According to the wide etiologic events, microcephaly may be classified in different ways: primary (congenital) or secondary (postnatal); isolated (affecting exclusively cerebral development); or syndromic (associated with extracranial anomalies and/or facial dysmorphisms). ^{5 6 7}

In familial microcephaly, the brain anomaly may be inherited in an autosomal dominant, recessive, or X-linked fashion. Microcephaly primary hereditary (MCPH) is an uncommon disorder, mainly presenting with autosomal recessive inheritance. The prevalence of MCPH is presumed to range from 1:30,000 to 1:250,000 live births. ⁷ Recent data state an incidence ranging from 1.3–150/100,000 in relationship to the level of consanguinity registered in the population. ²

MCPH individuals present with normal intelligence or variable intellectual disability according to the grade of OFC reduction. ^{8 9} Several genes have been related to the MCPH after the result of the first report in 1998 by Jackson et al ⁹ showed that MCPH was mapping to chromosome 8p22-pter. Eighteen OMIM genes have been related to MCPH and this number is likely to increase progressively in a short period of time. ¹⁰ Biallelic mutations in ASPM are reported as the most frequent cause of MCPH followed by mutations in the WDR62 gene and MCPH1 gene. ^{11 12 13 14}

Hashimoto's thyroiditis (HT) is a chronic autoimmune thyroid disorder, mainly affecting females, and related to the production of antithyroid antibodies with an infiltration of hematopoietic mononuclear cells, mainly autoreactive B and T lymphocytes in the interstitium among the thyroid follicles. ¹⁵ In this disorder, dysfunction of the endocrine gland may be clinically silent or manifest with subclinical or clear signs of hypothyroidism. The term HT includes, beyond the classic type, other clinical pathologic entities: fibrous variant, immunoglobulin G4-related variant, juvenile form, hashitoxicosis, and painless thyroiditis (sporadic or postpartum). ^{16 17}

We reported on twin female children affected by primary microcephaly with an OFC of −2 or −3 SD and a mild to moderate intellectual disability. Genetic analysis disclosed a heterozygous novel missense variant of the gene MCPH1 in both the twins. At the age of 10 years, the twins exhibited signs of thyroid dysfunction and a diagnosis of autoimmune juvenile thyroiditis was made.

Here, we discuss two main points: (1) the possible role of the heterozygous novel MCPH1 variant on the pathogenesis of microcephaly in the twins and (2) the unusual onset of the thyroid dysfunction in a short interval of time in both twins.

Case Report

These 11-year-old mono-ovular twins were born by cesarean section from indigent parents of Italian origin. A few days after their birth, the twins were adopted by a Sicilian family, who were already parents of three legitimate sons. Their small head was noticed since the first day of life and their developmental stages were reached with a slight delay. The first year of their life passed normally with regular episodes of infectious diseases. Nursery and primary schools were regularly followed up but the twins needed extra support at school. At the age of 10 years, due to slow growth, episodes of emotive liability and asthenia, the girls were submitted to several laboratory analyses, including endocrine assessment and thyroid investigations, that disclosed a high value of antithyroid peroxidase (anti-TPO), leading to a diagnosis of autoimmune juvenile thyroiditis with normal thyroid function. No treatment was started. One year later, the twins came to our observation for general control.

Twin 1

At physical examination, twin 1 presented in good condition. Her weight was 25 kg (3rd percentile), height 136 cm (25th percentile), and OFC 48 cm (−2 or −3 SD). She showed not impressive facial dysmorphism, consisting of thick hair and eyebrows, receding forehead, protruding and large ears, bulbous nose, and thin lips. The breast development was in Tanner stage 2, and pubic hairs were slightly pigmented. No axillary hair was noticed. The feet were long, with a large big toe. Teeth, heart, thorax and abdomen, and genital organs were normal. She attended elementary school with mild insufficient performance and need of support. Wechsler Intelligence Scale for Children, Third Edition (WISC III) IQ is 60 (n.v. 80–120). Neurological examination showed normal patellar reflexes and normal muscle tone and strength. No seizures were recorded.

Twin 2

As her twin, this patient presented in good condition. Her weight was 24 kg (3rd percentile), height 134 cm (10th percentile), and OFC 47.5 cm (−2 or −3 SD). The facial dysmorphism was similar to her twin with thick hair and eyebrows, receding forehead, protruding and large ears, bulbous nose, and thin lips. The breast development was in Tanner stage 2, pubic hairs were present with no axillary hair. Teeth, heart, thorax and abdomen, and genital organs were normal. The primary school teacher referred that she is a diligent, pacific, quite girl with mild difficulties in learning. Her IQ was calculated to be 58 (WISC III). No seizures were reported.

In both the patients, laboratory analyses were normal, including blood count, coagulation testing, blood lactate, pyruvate, glucose and ketones, creatinine kinase, copper, and ceruloplasmin. Plasma and urine amino acids, urinary organic acids, hemoglobin electrophoresis, purine and pyrimidines, plasma 7-dehydrocholesterol, and total cholesterol were also normal. Audiometric examination, electrocardiogram, and echocardiogram were normal. Video electroencephalogram performed while awake and during sleep showed no anomalies. Kidney and abdominal ultrasonography were normal. Thyroid ultrasound showed echostructure of the two lobes strongly inhomogeneous with small and diffuse hypoechoic areas in both the lobes and isthmus. Volume was calculated in 4.45 cm (+ 0.52 SD).

Magnetic resonance imaging (MRI) of the brain in twin 1 performed at the age of 2 years showed small brain, particularly in the frontal lobe, with thin smooth cortex and normal corpus callosum ( Fig. 1A , 1B )

Fig. 1.

( A, B ) Magnetic resonance imaging of the brain of the twin 1 at the age of 2 year with the classical aspect of microcephaly.

We followed up the twins for 4 years. Physical examination was unchanged. Scholastic performance was slightly insufficient. In Table 1 , the results of thyroid function tests at the age of 10 to 12 years are reported.

Table 1. Results of thyroid function tests at the age 10 to 12 years.

Age, y	TSH (µIU/mL)	FT4 (pg/mL)	FT3 (pg/mL)	Ab-TPO	Ab-T
Twin 1
10	4.32	11.0	3.9	450.1 (<60)	>2,500 (<60)
10.5	2.03	10.4	7.8	96 (<9)	472 (<9)
11	1.1	9.6	6.0	374	752
12	2.08	10.6	8.2	276	408
Twin 2
10	6.77	10.6	4.20	2,139 (<60)	>2,000 (<60)
10.5	2.47	10.4	6.3	244 (<9)	288 (<4)
11	2.44	9.3	5.1	340	471
12	3.02	10.4	6.2	360	520

Abbreviations: Ab-T, thyroid antibody; Ab-TPO, thyroid peroxidase antibody; FT3, free tri-iodothyronine; FT4, free thyroxine; TSH, thyroid-stimulating hormone.

Methods

Blood samples of the twins were taken for DNA extraction and further molecular analysis. Technique used: Massive parallel sequencing on platform Ion Torrent PGM, Program Torrent Suite (Life Technologies); data analysis was performed with the program Ion Reported (Life Technologies). Panel used: Custom Panel Ion AmpliSeq (AD 101847) including 376 amplicons containing the exonic and intronic regions adjacent to the sites of splicing (coverage mean 99.64%). Uniformity of the running coverage was 96.59% with a coverage of 20 X for 99.72% and 100 X for 99.09% of the total tracts.

Confirmation technique: Sanger sequencing of products of amplification of DNA (ABI PRISM 3130XL Genetic Analyzer) and further data analysis with the program SeqScape v2.7 (Applied Biosystem) of tracts of the region codifying with minor coverage of 20 X and of tracts containing possible variants.

Specific Genetic Testing

The genes analyzed were the followings: MCPH1 NM_024596.3 (99.86%); MCPH2 / WDR62 NM_001083961.1 (100%); MCPH3 / CDK5RAP1 NM_018249.5 (100%); MCPH4 / CASC5 NM_170589.4 (100%); MCPH5 / ASPM NM_018136.4 (99.7%); MCPH6 / CENPJ NM_018451.4 (100%) MCPH7 / STIL NM_001048166.1 (100%); MCPH8 / CEP135 NM_025009.4 (99.92%); MCPH9 / CEP152 NM_001194998.1 (100%); MCPH10 / ZNF335 NM_022095.3 (97.79%); MCPH11 / PHC1 NM_004426.2 (97.66%); MCPH12 / CDK6 NM_001259.6 (100%).

Results

Variants identified: MCPH1 ; chr 8:6357416; c.2180 C > T (rs199861426);p.Pro727Leu; heterozygous; missense; variant of uncertain significance (class 3).

Discussion

The main clinical features presented by the twins consisted of primary microcephaly (MCPH) with OFC measuring −2 or −3 SD, facial dysmorphism typical of nonsyndromic microcephaly, and cognitive development, slightly insufficient with a modest but regular scholastic performance and extra support at school. The absence of malformations and anomalies at brain MRI, negative analyses of congenital infections, and inborn error of metabolism agreed with the diagnosis of primary MCPH. Neither seizures nor frequent infectious episodes were recorded. With the exception of poor staturo-ponderal growth, the clinical course of the twins passed normally. At the age of 10 years, the twins started to have, in coincidental time, marked asthenia and emotive episodes and laboratory exams disclosed a high level of anti-TPO, leading to the diagnosis of autoimmune juvenile thyroiditis with normal thyroid function.

Primary microcephaly (also called “microcephaly vera”) refers to congenital abnormal cerebral growth, more frequently transmitted as an autosomal recessive tract, not associated with abnormalities of the other organs and with a not progressive course. ^{2 5} The affected patients may show variable cognitive disability usually correlated with the OFC: the smaller the OFC, the more severe appears to be the intellectual disability. In a small percentage of patients with −2 or −3 SD, a normal cognitive development may be kept on. ^{3 4}

Clinical diagnosis of microcephaly may be made at a glance, but a definite etiologic diagnosis is not easy, considering the several disorders which may present with this anomaly. In a retrospective study, performed on a cohort of 680 children affected by microcephaly, putative etiological diagnosis was obtained in 59% of the patients, but in the remaining 41%, no definitive diagnosis was established.

In this group of patients, a genetic cause was recognized in about half of the patients, perinatal and postnatal brain damage in 45 and 3% of cases, respectively. ¹⁸

Medical history, detailed clinical examination, MRI of the brain, ophthalmologic investigation, and screening for inborn errors of metabolism including phenylketonuria in maternal blood are mostly indicated to get the diagnosis. A relevant help in the diagnosis of microcephalic children has to be linked to the use of genetic investigations including array comparative genomic hybridization, Sanger sequencing of selected genes, or next-generation panel sequencing.

In the twins, on the basis of clinical and laboratory investigations, the etiologic diagnosis was circumscribed to monogenic cause of microcephaly among the group of autosomal recessive primary microcephaly, and the differential diagnosis was made with other disorders and/or syndromes in which microcephaly is the presenting sign, including Seckel's syndrome (microcephalic dwarfism type 1), ^{19 20} the α thalassemia mental retardation X-linked syndrome, ^{21 22} and the Nijmegen breakage syndrome. ^{23 24} Several genes and protein products have been identified in patients with autosomal recessive primary microcephaly. Eighteen MCPH loci (MCPH1_MCPH18) were mapped and contain the following genes: Microcephalin , WDR62 , CDK5RAP2 , CASC5 , ASPM , CENPJ , STIL , CEP135 , ZNF335 , PHC1 , CDK6 , CENPE , SASS6 , MFSD2A , ANKLE2 , CIT , and WDFY3 . ²⁵ All these genes play a relevant role in centriole biology, mitosis, plane, and abnormal chromatin modifications. Recently, in a mutant mouse model, a mitotic delay in the neuronal progeny causing increased apoptosis has been reported as a possible etiopathogenetic factor in primary microcephaly. ¹⁸ MCPH1 gene has the function to regulate cell progression into mitosis. The gene is located on chromosome 8p23.1 and its mutation is cause, among the others, of anomalous regulation of chromosome condensation, microtubule dynamics, and increased frequency of prophase-like cells. ^{18 25 26 27 28 29} MCPH1 dysfunction acts in increasing the production of early-born neurons of the deep layers (IV–VI) and decreasing the late-born neurons involved in the production of the thinner outer cortex layers (II and III). ^{30 31 32}

In the twins, a genetic panel performed for diagnostic evaluation for genes known to be associated or to be potentially associated with autosomal recessive primary microcephaly disclosed a heterozygous missense in MCPH1 gene with type of variant not yet reported. As known, the cases of MCPH are reported to be linked to homozygous or heterozygous compound events differently to what has been observed in these twins. Moreover, in recent databases, the molecular variant found in the twins has been classified as of uncertain significance and therefore cannot alone explain the cerebral anomaly manifested by the twins. However, we have hypothesized that the novel mutation involving the MCPH1 gene may have acted in some way causing the partial brain arrest. Recently, Naseer et al ³³ reported on a large family, in which two members were affected by primary microcephaly. Here, the genetic analysis disclosed two missense variants (c.982G > A) and (c.1273 T > A) in heterozygous state in exon 8 of the MCPH1 gene.

Both the patients, 5 and 10 years old, showed an OFC of −6 and −5 SD, respectively, with severe intellectual disability and no other anomalies as is reported in the cases of MCPH1-related primary microcephaly in which the small head is usually not associated with other malformation anomalies. ³³ Microcephaly has been found in patients with a heterozygous p.Thr258Met mutation in the KIF1A gene in a Korean family in which hereditary spastic paraplegia was associated with several other features, including intellectual disability, language delay, epilepsy, optic nerve atrophy, thinning of corpus callosum, and periventricular white matter lesion. ³⁴ Two patients with novel, de novo dominant missense mutations in KIFIA have been reported presenting with clinical features of severe hereditary spastic paraplegia and complicate progressive central nervous system involvement. ³⁵ We maintain that the novel heterozygous variant in the MCPH1 gene, found in the twins, may have contributed directly or indirectly in causing the microcephaly. Other factors in association with the heterozygous novel variant, including epigenetic events, may have a role in causing the impaired cerebral development manifested by the twins. Further similar observations may confirm this hypothesis.

In the twins, another aspect to highlight is the onset of the thyroid disorder in a coincident period of time. Due to signs of asthenia, tiredness, slow growth, and behavioral disturbances, manifested by the twins at the same time and the subsequent analysis of high value of anti-TPO, a diagnosis of autoimmune juvenile thyroiditis was performed. The association of primary microcephaly and onset of autoimmune thyroiditis in juvenile age may be casual. On the contrary, to our knowledge, no cases of primary microcephaly in association with this thyroid disorder have been reported. Much more intriguing is the autoimmune juvenile thyroiditis with onset in a short period of time in both the twins. The anti-TPO and atrophic autoimmune thyroiditis findings are expressions of a specific immune response directed against thyroid structures of self and it is well known that there is an interaction between genetic susceptibility and environmental factors in the pathogenesis of autoimmune juvenile thyroiditis. Studies in cells and tissues reported in individuals affected by autoimmune thyroid disease have shown that epigenetic influences in these disorders may interfere with the normal regulation of gene expression. ^{15 16} Other reports have confirmed the thesis that immune defects in groups of patients may raise from environmental factors in individuals with genetic susceptibility. ^{15 16 36 37} To confirm the familiar predisposition of HT, there is a recent study of Kust and Matesa ³⁸ conducted in 39 patients with a positive family history of HT, in comparison with a control group of affected patients without family history of the disorder: family positive history was found in 17 patients compared with 7 of the control group ( p  = 0.0262), confirming the genetic predisposition of HT. The coincidence of the onset of the thyroid disorder in the twins is difficult to explain. We can advance the hypothesis that a time-related, preprogrammed molecular/cellular event may have acted triggering the disorder. A second hypothesis is that environmental triggers may act at the same time in the genetically predisposed twins.

In conclusion, the report of these twins seems to be worthwhile since the novel heterozygous missense MCPH1 variant may be an example as it may have directly or indirectly acted with other genes or with epigenetic factors in causing the brain impairment. Interesting is also the observation of the onset of autoimmune juvenile thyroiditis, manifested in a so short period of time in the twins showing the genetic predisposition in the pathogenesis of this disorder. Further observations on this topic are useful in confirming how we have learned by this clinical experience.

Funding Statement

Funding None.